Plant promoter and method for gene expression using said promoter

ABSTRACT

A plant promoter capable of inducing the expression specifically at the site and stage wherein the reconstitution of plant cell wall xyloglucan is necessary, namely, a plant promoter originating in a gene which encodes an endo-xyloglucan transferase or a gene which encodes a substance having a function equivalent thereto; and a method for modifying the function of a plant with the use of the plant promoter and a method for cloning the plant promoter.

FIELD OF THE INVENTION

The present invention relates to plant promoters which are useful fordevelopment of new plant varieties employing the gene recombinationtechnology and the plant engineering such as functional modificationetc. as well as is useful for the plant cell engineering such asfunctional modification of plant culture cells producing usefulmetabolites, DNA fragments in which useful genes are ligated to thepromoters in such state that the useful genes can be expressed, andvectors containing the DNA fragments. Furthermore, the present inventionrelates to plants or plant cells that are transformed with the DNAfragments or vectors containing the DNA fragments, or to transgenicplants regenerated from the plant cells. Still furthermore, the presentinvention relates to a method for cloning the plant promoters.

PRIOR ART

Improvement of plants utilizing the gene engineering techniques hasrecently been using practically [Science, 244, 1293-1299 (1989)]. Inparticular, remarkable able progress has been made in the transformationsystem utilizing Ti plasmid and Ri plasmid that are contained in soilbacteria, Agrobacterium tumefaciens and Agrobacterium rhizogenes,whereby the system can be applicable not only to tobacco, Arabidopsis,and petunia that have been hitherto transformed but also todicotyledonous plants such as azuki bean [Abstracts of Presentation atthe Yeeting of NIHON SHOKUBUTU SOSHIKIBAIYOU GAKKAI (JapaneseAssociation for Plant Tissue Culture), P. 124 (1990)] and tomonocotyledonous plants such as rice [The Plant Journal, 6, 271-282(1994)]. Moreover, for monocotyledonous plants whose representativeexample is the rice plant, a method comprising preparing a protoplastand then transferring a gene therein by electroporation has beenpractically used [Nature, 338, 274-276 (1989)]. In addition, there aremany examples where genes are directly transferred into plants using theparticle gun method [The Plant Journal, 2, 275-281 (1992)].

As for promoters which induce the tissue-specific expression of usefulsubstances or enzymes, there have been heretofore isolated genes thatexpress specifically in respective tissues of seed [SHOKUBUTU SAIBOUKOUGAKU (Plant Cell Technology), 3, 568-576 (1991)], respective tissuesof leaves and flowers [Science, 250, 931-936 (1990)], tuber [SHOKUBUTUSAIBOU KOUGAKU (Plant Cell Technology), 3, 577-587 (1991)], tuberousroot, and root nodule [Science, 250, 948-954 (1990)) and the expressionby these promoters has been analyzed in transgenic plants.

However, most promoters that have been hitherto utilized for thesevector systems are promoters originating from Ti plasmid contained inAgrobacterium tumefaciens and promoters originating from the genes ofcauliflower mosaic virus (CaMV). These promoters constitutively expressirrespective of growth stages and tissues of transgenic plants and cannot be controlled. In addition, the expression level is low. Moreover,among promoters containing expression regulatory regions inducing thetissue-specific expression, none of the promoters induce the expressionspecifically at the site and the stage required for the reconstitutionof plant cell wall xyloglucan.

Furthermore, in the field of plant cell engineering, even when oneintends to produce a useful secondary metabolite in plant cells to beused for a plant tissue culture, there have been known many cases wherethe expression of an enzyme gene in a biosynthesis system of themetabolite is repressed due to the presence of a plant hormone essentialfor the cell growth, thereby repressing the production of the metabolite[Physiologia Plantarur, 80, 379-387 (1990)]. Therefore, it is extremelydifficult to optimize the biosynthesis of a secondary metabolite bycells in the presence of a plant hormone necessary for the cell growth.Then, it is required to employ a two-stage culture method wherein thecell growth and the biosynthesis of a secondary metabolite are carriedout under separate conditions [Nippon NOUGEIKAGAKU KAISHI (Journal ofAgricultural Chemistry Society of Japan), 60, 849-854 (1986)).

Such repression can be considered to be caused by regulation of thepromoter of an enzyme gene in a biosynthesis system by a signal from aplant hormone and the like to repress the expression.

Accordingly, it is considered that the biosynthesis of a secondarymetabolite can be facilitated under cell growth conditions bytransferring a chimeric gene, in which the above promoter is replaced bya promoter inducing the expression of a useful substance or enzymeabundantly during a cell growth period, into cells. Nevertheless, anypromoter abundantly inducing the expression of a useful substance orenzyme especially during a cell growth period has not been known in thefield. Therefore, if such a promoter abundantly inducing the expressionof a useful substance or enzyme especially during a cell growth periodis available, the biosynthesis of a secondary metabolite can be effectedas cells grow and significant improvement in the productivity of theuseful secondary metabolite can be expected.

Thus, promoters that can induce the tissue specific expression or thatcan control the expression with a plant hormone and the like have beendesired in the plant engineering and the plant cell engineering.

OBJECTS OF THE INVENTION

The object of the present invention is to provide a plant promoter thatcan induce the tissue-specific expression especially at the site and thestage required for the reconstitution of plant cell wall xyloglucan andfurther can control the expression with a plant hormone and the like, aDNA fragment containing the promoter, a vector containing the DNAfragment, a plant or plant cells transformed with the DNA fragment orvector, or a transgenic plant regenerated from the plant cells, and amethod for cloning the plant promoter.

SUMMARY OR THE INVENTION

The present inventors have directed their attention to the fact that theexpression of an endo-xyloglucan transferase (EXT) gene originating froma plant and its family genes is tissue-specific and have expected that apromoter that can control the expression and its vector would beavailable. The present inventors have further expected that suchpromoter can be utilized for improvement of plant cells and plants.Then, the present inventors attempted to clone a region containing apromoter which was presumed to be located upstream from a plant EXTgene. However, it was difficult to clone the promoter of the EXT gene bya known plaque hybridization method because of the presence of manyfamily genes including pseudogenes and a decrease in the plaque-formingability of plaques obtained by preparing a phage having a fragmentcontaining a region located in upstream from the EXT gene and its familygenes and infecting a host with it.

Then, the present inventors have studied intensively. As a result, thepresent inventors have succeeded in cloning the promoter of the EXT geneand analyzed the promoter portion to determine its nucleotide sequence.This promoter portion was cleaved off and ligated to β-glucuronidase(GUS) gene originating from E. coli and the resultant chimeric gene wastransferred into plant cells.

It has been confirmed that GUS gene is intensely expressed in the cellsinto which the gene was transferred. When, according to the same manner,nucleotide sequences of the promoter portions of family genes of EXTgene were determined and ligated to GUS gene originating in E. coli andthe resultant chimeric gene was transferred into plant cells, theintense expression of GUS gene was also confirmed.

Furthermore, it has been confirmed by northern hybridization that theEXT gene containing this promoter is expressed in a tissue-specificmanner especially at the site and the stage required for thereconstitution of plant cell wall xyloglucan and that there exists eachof the EXT gene and its family genes containing the promoter which isexpressed during the logarithmic growth phase or the stationary phase ofculture cells. Thus, the present invention has been completed.

That is, in brief, the first aspect of the present invention relates toa plant promoter inducing the tissue-specific expression and ischaracterized in that the plant promoter can control the expression of agene encoding an enzyme having the function to carry out thereconstitution of plant cell wall xyloglucan and, particularly, it has apromoter activity at the site required for the reconstitution of plantcell wall xyloglucan or has a promoter activity at the stage requiredfor the reconstitution of plant cell wall xyloglucan.

The second aspect of the present invention relates to a plant promoterof the first aspect of the present invention and is characterized inthat it is contained in any nucleotide sequence selected from SEQ ID NO1, 2, 3, 4, 5, 6, 7, and 8 in the Sequence Listing.

The third aspect of the present invention relates to a plant promoter ofthe first aspect of the present invention and is characterized in thatit is hybridizable to the nucleotide sequence in the second aspect andhas a promoter activity in plants or plant cells, or in transgenicplants regenerated from the plant cells.

The fourth aspect of the present invention relates to a DNA fragmentcontaining the plant promoter of the first, second or third aspect andis characterized in that it is ligated to the plant promoter in a statecapable of expressing a useful gene.

The fifth aspect of the present invention relates to a vector and ischaracterized in that it contains the plant promoter of the first,second or third aspect or the DNA fragment of the fourth aspect.

The sixth aspect of the present invention relates to a plant or plantcells transformed with the DNA fragment of the fourth aspect or thevector of the fifth aspect. mode, or to transgenic plants regeneratedfrom the plant cells.

The seventh aspect of the present invention relates to a method forproducing protein characterized in that at least one of the transformedplant and plant cells and transgenic plants regenerated from the plantcells of the sixth aspect is used.

The eighth aspect of the present invention related to a method forcontrolling morphology of a plant and is characterized in that the DNAfragment of the fourth aspect or the vector of the fifth aspect is used.

The ninth aspect of the present invention relates to a method forcloning a plant promoter and is characterized in that a gene encoding anenzyme having the function to carry out the reconstitution of plant cellwall xyloglucan and, particularly, a gene encoding endo-xyloglucantransferase or its functional equivalent is used.

DETAILED EXPLANATION OF THE INVENTION

The "promoter" used herein contains a TATA box. region or TATA-box likeregions which are located 20 to 30 base pairs upstream from thetranscription initiation site (+1) and are responsible for initiation ofthe transcription by an RNA polymerase from an exact position. However,it is not necessarily limited to in front and behind these regions andmay contain any other region which is required for association of aprotein other than a RNA polymerase for regulation of the expression inaddition to the above regions.

And, sometimes, the term "promoter region" is used in the presentspecification and this means a region. containing the promoter asdescribed in the present specification.

The "promoter activity" used herein means the ability and function toproduce a gene product of a useful gene outside or inside a host (aplant, plant cells or a transgenic plant regenerated from the plantcells), when the useful gene is ligated to a site downstream from apromoter in order to express and then the resultant gene is transferredinto the host.

In general, the promoter activity is indicated as positive or negative,or strong or weak, by ligating a gene encoding a protein capable of easyassay (a reporter gene) to a site downstream from a promoter in order toexpress, transferring the resulting promoter into the host, and thenmeasuring the expression level of the protein. Thus, when a useful geneis ligated to a site downstream from a promoter in order to express andthen the resultant gene is transferred into a host, the confirmation ofexpression of a gene product of the useful gene outside or inside thehost shows that the promoter has the promoter activity in thetransferred host.

The phrase "a gene encoding an enzyme having the function to carry outthe reconstitution of plant cell wall xyloglucan" used herein means agene encoding an enzyme specifically expressed in the reconstitution ofplant cell wall xyloglucan and, particularly, refers to a gene encodingendo-xyloglucan transferase (EXT) and family genes of EXT gene. Examplesof family genes of EXT gene include BRUl gene [Plant Physiology, 104,161-170 (1994)], meri-5 gene [The Plant Cell, 3, 359-370 (1991)], andXRP gene obtained in the present invention.

The phrase "a site required for the reconstitution of plant cell wallxyloglucan" used herein means a site where a gene encoding an enzymehaving the function to carry out the reconstitution of plant cell wallxyloglucan is expressed specifically and, in so far as the gene encodingthe enzyme having the function to carry out the reconstitution of plantcell wall xyloglucan is expressed specifically, the site where theexpression occurs is included in the site required for thereconstitution of plant cell wall xyloglucan as mentioned herein.

For example, sometimes, the specifically expression site of EXT genewhich is one of genes encoding an enzyme having the function to carryout the reconstitution of plant cell wall xyloglucan differs from thoseof family genes of EXT gene even in the same plant. However, all of themare included in the site required for the reconstitution of plant cellwall xyloglucan as mentioned in the present specification.

The stage of plant growth for the reconstitution of plant cell wallxyloglucan" used herein means the stage when the gene encoding theenzyme having the function to carry out the reconstitution of plant cellwall xyloglucan is expressed specifically and, in so far as the geneencoding the enzyme having the function to carry out the reconstitutionof plant cell wall xyloglucan is expressed specifically, the stage whenthe expression occurs is included in that required for thereconstitution of plant cell wall xyloglucan as mentioned herein.

For example, sometimes, the specific stage of EXT gene expression whichis one of genes encoding an enzyme having the function to carry out thereconstitution of plant cell wall xyloglucan differs from those offamily genes of EXT gene even in the same plant. However, all of themare included in the stage required for the reconstitution of plant cellwall xyloglucan as mentioned in the present specification.

For example, in culture cells, mitotic cells are abundant in thelogarithmic growth phase and the synthesis and reconstitution of thecell wall are vigorously carried out. In the stationary phase, cellselongate actively and thereby the reconstitution of cell wall isrequired. Thus, the cell wall reconstitution is required in both phases.As described in Example 10 hereinafter, the expression stage in theculture cells for EXT gene originating from tobacco is completelydifferent from that for the XRT gene, a family gene of EXT geneoriginating from tobacco. In other words, EXT gene originating fromtobacco is intensely expressed specifically in the logarithmic phase,whereas the expression in the stationary phase is reduced to about onetwentieth. In contrast, XRT gene, a family gene of EXT gene originatingfrom tobacco, is intensely expressed specifically in the stationaryphase. Thus, the stage for specific expression of enzymes exhibiting thesame enzymatic activity is controlled by the promoter of the geneencoding the enzyme. Examples of the stage for the reconstitution ofplant cell wall xyloglucan as mentioned herein also include thelogarithmic phase and the stationary phase in such cases.

The term "a functional equivalent" used herein means as follows.

A naturally occurring protein is subject to various mutations in itsamino acid sequences such as deletion, insertion, addition, andsubstitution of the amino acid(s) by modifications of the formed proteinoccurring in the living body and during the purification process, inaddition to polymorphism and mutations of the gene encoding the protein.Nevertheless, there has been known the existence of a molecule thatexhibits a physiological and biological activity substantiallyequivalent to that of the protein with no mutation. Such a molecule thathas a different structure but possesses a substantially equivalentfunction is defined as a functional equivalent.

The same is true in the case where the above-mentioned mutations areartificially introduced into the amino acid sequence of protein. Avariety of mutants prepared in such cases can be interpreted asfunctional equivalents in so far as they exhibit a biological activitysubstantially equivalent to that of the protein with no mutation.

For example, it is said that methionine residue existing in theN-terminus of protein expressed by E. coli is removed in many cases bythe action of methionine aminopeptidase. However, sometimes, bothproteins with and without methionine residue are formed depending on aparticular type of protein. Nevertheless, the presence or absence ofmethionine residue does not influence the activity of the proteins inmany cases. In addition, it has been known that a polypeptide obtainedby replacing certain cysteine residue of interleukin-2 (IL-2) withserine residue maintains interleukin-2 activity [Science, 224,1431-1433].

Furthermore, in the protein production by the gene engineering, proteinis often expressed as fusion protein. For example, an N-terminal peptidechain derived from another protein is added to the N-terminus of theobjective protein in order to increase an expression level of theobjective protein. And, an appropriate peptide chain is added to theN-terminus or the C-terminus of the objective protein in order tofacilitate purification of the objective protein by using a carrierhaving an affinity to the added peptide chain after the expression.

Moreover, it has been known that, for each of the amino acids in a gene,there are one to six codons (sets of three nucleotides) which define theparticular one amino acid. Accordingly, there can be many genes whichencode a particular amino acid sequence, though the number of the genesdepends on the amino acid sequence. Genes do not always exist stably inthe nature and mutations often occur on their nucleic acids. There is acase where a mutation occurring on a gene does not induce any change inthe encoded amino acid sequence (called as a silent mutation) and, insuch a case, it can be said that a different gene encoding the sameamino acid sequence is formed. Therefore, even if a gene encoding acertain defined amino acid sequence is isolated, a possibility can notbe denied that many types of genes encoding the same amino acid sequencemay be formed during the passage of the living organism containing thegene.

Furthermore, it is not so difficult to artificially prepare many typesof genes encoding the same amino acid sequence by employing a variety ofgene engineering techniques.

For example, in the protein production by the gene engineering, when acertain codon used in the inherent gene encoding the objective proteinis not frequently utilized in the host used, sometimes, a low expressionlevel is experienced. In such a case, an attempt has been made toincrease the expression of the objective protein by artificiallyreplacing the said codon to another codon which is more popular in thehost without influence on the encoded amino acid sequence. Needless tosay, it is quite possible to artificially prepare a variety of genesencoding a certain amino acid sequence in this way. Accordingly, eventhese artificially prepared different genes are included in the presentinvention, in so far as they code for the amino acid sequences that canbe deduced from the nucleotide sequences disclosed by the presentinvention.

Moreover, many of polypeptides, which undergo at least one ofmodifications of one or more amino acids by deletion, insertion,addition, and substitution in the amino acid sequence of the objectiveprotein, have an activity functionally equivalent to that of theobjective protein. Genes encoding such polypeptides are also included inthe functional equivalent of the present invention irrespective of beingisolated from nature or artificially prepared.

In general, in many cases, genes encoding the functional equivalentshave homology. Therefore, genes hybridizable to EXT gene used in thepresent invention and encoding a polypeptide having the same functionare also included in the functional equivalents of the presentinvention.

Examples of "a useful gene" mentioned herein include genes encodingproteins expressible in plants or plant cells or transgenic plantsregenerated from the plant cells, antisense RNAs of genes originatingfrom plants or plant cells or transgenic plants regenerated from theplant cells, genes encoding binding proteins of transcription factorsoriginating from plants or plant cells or transgenic plants regeneratedfrom the plant cells or decoys having sequences or analogous sequencesof binding sites for the transcription factors and ribozymes cleavingmRNAs originating from plants or plant cells or transgenic plantsregenerated from the plant cells.

Genes encoding proteins expressible in plants or plant cells ortransgenic plants regenerated from the plant cells are exemplified bythose originating from plants, but they are not limited thereto in thepresent invention and genes originating from microorganisms such asbacteria, yeasts, actinomycetes, fungi, ascomycotina, basidiomycotinaetc. and genes originating from living organisms such as animals etc.,as far as they can be expressed in plants or plant cells or transgenicplants regenerated from the plant cells.

"Decoys" mentioned herein are referred to DNAs genes encoding bindingproteins of transcription factors originating from plants or plant cellsor transgenic plants, regenerated from the plant cells or DNAs havingsequences or analogous sequences of binding site for the transcriptionfactors, which repress the action of the transcription factors upontransferring as "decoys" into cells.

"Ribozymes" mentioned herein are referred to molecules cleaving mRNAsfor defined proteins to inhibit the translation of these definedproteins. Ribozymes can be designed from gene sequences encoding definedproteins. Examples of ribozymes mentioned herein include any ribozymeswhich can cleave mRNAs for defined proteins to inhibit the translationof these defined proteins regardless of their types such ashammer-head-type ribozymes, hairpin-type ribozymes, delta-typeribozymes, etc.

In so far as a plant having an enzyme which functions to carry out thereconstitution of plant cell wall xyloglucan, any plant can be used inthe present invention. Examples of the plants include dicotyledonousplants such as azuki bean, soybean, Arabidopsis, tomato, potato,Brassica, sunflower, cotton, tobacco, etc. and monocotyledonous plantssuch as wheat, rice, corn, sugar cane etc., of which plants having EXTenzyme and EXT-analogous enzymes that are expressed in a tissue-specificmanner are particularly employed:

EXT gene is a housekeeping gene of plants and thereby many family genesexist. As for DNA fragments containing promoter regions of these familygenes, cloning of the promoter of EXT gene is not easy by a conventionalplaque hybridization method owing to the presence of many family genesincluding pseudogenes and decrease in the plaque-forming ability of theplaque obtained when a phage having a fragment of a upstream region fromEXT gene or its family genes is prepared and infected to a host.However, by overcoming these two problems, hybridization is applicableto any plants including dicotyledonous plants and monocotyledonousplants by employing cDNA of EXT gene and its family genes as a probe andgenomic DNA as a target, and also isolation is possible by investigatingPCR method in details.

As the probe, cDNA of EXT gene and its family genes of a plant differentfrom the target species can be used. However, it is preferred for moreeffective hybridization to select cDNA from a plant of the same speciesas the target as the probe. The present inventors have isolated cDNAs ofEXT gene from azuki bean (Vigna angularis), soybean (Glycine max),Arabidopsis (Arabidopsis, thaliana), tomato (Lycopersicon esculentum),wheat (Triticum aestivum), tobacco (Nicotiniana tabacum), rice (Oryzasativa), corn (Zea mays). Of these cDNA molecules, full-length orpartial nucleotide sequences for azuki bean, soybean, Arabidopsis,tomato, and wheat as well as the restriction map for rice and therestriction map for corn have been described in EP-0562836 A1 (1993) anda partial nucleotide sequence for tobacco has been described in JP7-79778 A. Also, partial nucleotide sequences for rice and corn areshown in SEQ ID NO 9 and SEQ ID NO 10 of the Sequence Listing.

The cDNAs of family genes can be isolated by using the full-length orpartial cDNA of EXT gene as a probe. For example, cDNAs of the familygenes can be isolated from a wide species of plants by using, as aprobe, a sequence conserved between all of the above-mentioned cDNAs ofEXT gene and a xyloglucanase gene of Tropaeolum majus [The PlantJournal, 3, 701-711 (1993)]. In addition, cDNAs of the family genes canbe isolated from a wide species of plants by synthesizing a primer onthe basis of the conserved region and then carrying out PCR [ConsensusPCR; Molecular and Cellular Biology, 13, 4745-4752 (1993)].

Hereinafter, the present invention is explained in details for azukibean as an illustration.

A cDNA library prepared by the method described in EP-0562836 A1 (1993)using seeds of azuki bean (WATANABE SHUSHI Co., Ltd.) can be utilizedfor searching clones transformed with family genes of EXT gene. The cDNAlibrary is prepared, for example, by preparation of RNAs from azukibean, followed by purification of poly(A)⁺ RNA using Oligotex-dT30(NIHON Roche Co., Ltd.) and then, for example, by treatment with areverse transcriptase using the poly(A)⁺ RNA and an oligo-dT primer toprepare cDNA. The cDNA library is prepared from the cDNA by using cDNASynthesis Kit System Plus (Amersham). This cDNA library is utilized forplaque hybridization using the cDNA of EXT gene as a probe to obtain,for example, 96 positive plaques selected from 5×10⁴ plaques. Theseplaques are amplified by the plate lysate method (T. Maniatis et al.,Molecular Cloning, A laboratory Manual, Second Edition, Chapter 2, pp.60-66, published by Cold Spring Harbor Laboratory Press in 1989),followed by dot hybridization to classify the plaques on the basis ofthe signal strength.

Phages are isolated from two plaques indicating the signal strengthdifferent from that of EXT gene of azuki bean and DNAs inserted thereinare extracted. These DNAS are cleaved with restriction enzyme EcoR I(TAKARA SHUZO Co., Ltd.) and the lengths of DNA fragments are identifiedby agarose gel electrophoresis. The identifiers DNA fragments of about730 bp, 430 bp, and 1090 bp are purified and subcloned at the EcoR Isite of pUC18 (TAKARA SHUZO Co., Ltd.). The resulting plasmids are namedas pVX44-1, pVX44-2, and pVX45-1, respectively. These plasmids areemployed for determination of the nucleotide sequences of the DNAfragments according to the Sanger method using BcaBEST™ DideoxySequencing Kit (TAKARA SHUZO Co., Ltd.), indicating that two genes of ahigh homology with EXT gene (azuki bean EXT) are cloned. Parts of theirnucleotide sequences are shown in SEQ ID NO 11 and SEQ ID NO 12 in theSequence Listing (azuki bean EXT 2 and azuki bean EXT 3).

The above-mentioned cDNA library is utilized for plaque hybridizationusing one of the above-mentioned conserved sequences (SEQ ID NO 13) as aprobe to obtain, for example, 8 positive plaques searched from 8×10³plaques. DNAs inserted in phage vectors of the plaques are extractedand, for example, a DNA fragment (about 1.2 kbp) of a high homology withEXT gene as well as with its family genes, the BRUl gene [PlantPhysiology, 104, 161-170 (1994)] and the meri-5 gene [the Plant Cell, 3,359-370 (1991)] can be obtained. A part of this DNA nucleotide sequenceis shown in SEQ ID NO 14 in the Sequence Listing (azuki bean XRP 1).

Furthermore, the above-mentioned cDNA library can be utilized, forexample, for PCR using the above-mentioned, conserved sequence andoligo-dT primer. As a result, a DNA fragment of a family gene differentfrom, for example, azuki bean EXT, azuki bean EXT 2, azuki bean EXT 3,and azuki bean XRP 1 can be obtained. A part of this DNA nucleotidesequence is shown in SEQ ID NO 15 in the Sequence Listing (azuki beanXRP 2).

In a plant other than azuki bean, plaque hybridization using one of theabove-mentioned conserved sequences (SEQ ID NO 13) as a probe can beutilized to obtain a cDNA of a family gene. For example, a commerciallyavailable cDNA library of tobacco is utilized for plaque hybridizationusing one of the above-mentioned conserved sequences (SEQ ID NO 13) as aprobe to obtain 30 positive. plaques searched from about 3×10⁴ plaques.DNAs inserted in phage vectors of said plaques are extracted and, forexample, a DNA fragment (about 1.2 kbp) of a high homology with EXT geneas well as with the BRUl gene (Plant Physiology, 104, 161-170 (1994)]and the meri-5 gene [the Plant Cell, 3, 359-370 (1991)] can be obtained.A part of this DNA nucleotide sequence is shown in SEQ ID NO 16 in theSequence Listing (tobacco XRP 1).

Next, for example, a genome DNA library of azuki bean can be obtained bypreparing a genome DNA from the leaves of azuki bean by a conventionalmethod, subjecting it to partial digestion using restriction enzymeSau3A I, subjecting the partial digestion product, for example, toligation to a vector λGEM-11 using λGEM-11 Xho I Half-Site Arms CloningSystem (Promega Biotec) followed by packaging using an in vitropackaging kit (Stratagene), and then infecting the resulting fragment toa host. This library can be utilized, for example, for hybridizationusing a cDNA of EXT gene described in EP-0562836 A1 (1993) as a probe tosearch phages having a DNA fragment containing a promoter region of thisgene. For example, 10 positive plaques are obtained from about 1×10⁵plaques and DNAs inserted in phage vectors of the plaques are extractedto obtain DNA fragments of an average length of about 15 kbp. These DNAfragments are utilized for Southern hybridization using a DNA fragmentcontaining a cDNA of EXT gene as a probe followed by subcloning theobjective fragment to a plasmid vector to analyze a partial nucleotidesequence. As a result, for example, it can be confirmed that DNA.fragments inserted in phage vectors of all plaques have a sequenceanalogous to EXT gene.

These studies may suggest that cloning of regions containing promotersof EXT gene and its family genes can be easily carried out. However, infact, the following two problems were caused and cloning of any promoterof EXT gene by a conventional plaque hybridization was failed.

The first problem is the existence of many family genes includingpseudogenes that are not easily differentiated by conventionalhybridization. Accordingly, in order to carry out cloning of genomic DNAclones that are true counterparts of the objective cDNA clones, it isnecessary to analyze and determine all the nucleotide sequences ofrespective genomic DNAs after roughly screening genomic DNA clones thatmay be the counterparts. Alternatively, it is necessary to find out anucleotide sequence that can distinguish respective genes in familygenes by analyzing many family genes from cDNAs and then to carry outhybridization using its nucleotide sequence-specific oligo probe (SSOP)to define the genomic DNA clone.

The second problem involves a strong repressing action that is inducedon transfer of DNA fragments containing promoter regions of EXT gene andits family genes as well as decrease in the plaque-forming ability thatoccurs on infection of phages in a host bacterium (E. coli). In fact, itis difficult to search for the above-defined phages containing promoterregions of EXT gene because formed plaques are extremely small ascompared with normal phages. Such a problem has been revealed first inthe course of repeated trial and error to isolate the objective promoterregion and is quite unpredictable until cloning is actually carried out.

Then, the present inventors have intensive studied to solve theabove-mentioned problems, resulting in the first successful cloning of aregion containing the promoter of EXT gene, after steadily solving theproblems one by one by utilizing a variety of gene engineeringtechniques including an improved PCR method.

Hereinafter, EXT gene and its family genes are collectively referred toas EXT-family genes in the following explanations.

Cloning of Promoter

In case where an influence by the above-mentioned repression of theplaque-forming activity exists, it is impossible to clone a promoterregion of EXT gene, even after repeated screening from entire genomicDNA libraries. Then, it is conceivable to prepare short genomicfragments that are not susceptible to the repression and then carry outcloning under a minimized influence of the repression. For this purpose,it is required to subject genomic DNAS, first, to complete digestionwith a variety of restriction enzymes followed by genomic Southernhybridization and then to deduce what size of DNA fragment having theterminal site of which restriction enzyme contains the objectivepromoter region of EXT gene.

A partial genomic DNA library of the thus-defined DNA size can beprepared by complete digestion of genomic DNAs with the thus-definedrestriction enzyme and by recovering, from agarose gel, peripheral DNAfragments having the defined DNA-fragment size as an average.

As a result, a partial genomic DNA library that is condensed about morethan ten-fold as compared with the original entire genomic DNA librarycan be obtained. For example, genomic DNAs prepared from the leaves ofazuki bean by a conventional method are subjected to digestion with, forexample, restriction enzymes BamH I, EcoR I, and Hind III (all: TAKARASHUZO Co., Ltd.), followed by genomic Southern hybridization to indicatethe formation of 2 or 3 bands, wherein the most intense band can beidentified at more than 15 kbp by the BamH I digestion, at about 8.5 kbpby the EcoR I digestion, at about 8.5 kbp by the Hind III digestion, andat about 5.5 kbp by the EcoR I-Hind III double digestion.

Of these bands, the band identified at about 5.5 kbp by the EcoR I-HindIII double digestion is recovered from the agarose gel and subjected toligation, for example, to the EcoR I and Hind III sites of λEXlox(Novagen), followed by packaging by an in vitro packaging kit(Stratagene) and infection to a host bacterium, thereby enabling toobtain a partial genomic DNA library with DNA fragments of an about 5.5kbp size as an average that has the EcoR I and Hind III sites at bothterminals and is more condensed as compared with the entire genomic DNAlibrary. This condensed library is subjected to hybridization using thecDNA of EXT gene as a probe as described above to search for a phagehaving a DNA fragment containing the promoter region of this gene. Forexample, 8 positive plaques searched from 1.3×10⁵ plaques are subjectedto hybridization using an oligonucleotide VAN-U7 (SEQ ID NO 17),synthesized on the basis of a 5'-uncoded region of a lower homology withcDNAs of family genes other than EXT gene, as a probe, therebyconfirming that, for example, 4 out of 8 plaques are DNA fragmentscontaining the cDNA of EXT gene. λEXlox can be subjected to automaticsubcloning, since infection of this DNA fragment to a host bacteriumhaving the PlCre gene will convert a region subcloned automatically inthe host to a pUC-type plasmid by automatic subcloning.

DNA sequencing analysis using the plasmids inserted with the DNAfragment, followed by comparison of the DNA fragment with the cDNAsequence of EXT gene, identifies whether the DNA fragment contains apromoter of EXT gene.

Parts of DNA nucleotide sequence of the thus-identified fragment areshown in SEQ NO 18 (upstream from EXT coding region) and SEQ NO 19(downstream from the DNA fragment) in the Sequence Listing.

Hereinafter, a 5'-upstream from the EcoR I site is referred to as "thepromoter-upstream region" and a 3'-downstream as "thepromoter-downstream region", for convenience.

The plasmid integrated with the fragment is denoted as pVXG303, whereasE. coli JM109 strain transformed with pVXG303 is denoted and indicatedas Escherichia, coli JM109/pVXG303 and has been deposited on Mar. 15,1995 (the date of original deposit) at National Institute of Bioscienceand Human-Technology, Agency of Industrial Science and Technology,Ministry of Industrial Trade and Industry (1-3, Higashi 1-Chome,Tsukuba-Shi, Ibaragi-ken, 305, Japan) under the accession No. FERMBP-5390, in accordance with the Budapest Treaty.

The promoter-upstream region can be obtained by cloning of the Hind IIIfragment of about 8.5 kbp that is identified as mentioned above. Forthis purpose, genomic DNAs of azuki bean are completely digested withrestriction enzyme Hind III and then DNA fragments are recovered byseparation by 0.7% agarose gel electrophoresis, in the same manner asdescribed above. Also, complete digestion of λZAPII (Stratagene) withrestriction enzyme Spe I (TAKARA SHUZO Co., Ltd.), followed by thefill-in reaction in the presence of dCTP and dTTP, forms ahalf-filled-in site. The fill-in reaction of the Hind III fragment(average: about 8.5 kbp) in the presence of dATP and dGTP, as describedabove, followed by ligation into the half-filled-in site of λZAPII,enables a trial to prepare a genomic DNA library.

However, the size of this λDNA is marginal for the packaging and therebythe titer of its library is expected to be not so high. In fact, thetiter of this library is so low as to carry out screening effectively.This size is also too small to use a replacement-type phage vector suchas λDASHII (Stratagene). In fact, the titer of a library, which isobtained by ligation of the recovered Hind III fragment (average: about8.5 kbp) to the Hind III site of λDASHII is so low as to carry outscreening effectively.

In addition, the only method would be to carry out screening from theentire genomic DNA library using λGEM11 (Promega Biotec) by using thecDNA of EXT gene as a probe. However, it is expected that any fragmentcontaining the objective promoter of EXT gene could not be obtainedbecause of the influence of plaque-forming repression as well as theexistence of many family genes as described above.

Then, it is expected that the use of a newly cloned genomic fragment asa probe will result in a more intense hybridization by a fragmentcontaining the objective promoter than in the case using cDNA.Therefore, it is desirable to carry out plaque hybridization using sucha genomic DNA fragment as a probe and then screen plaques as many aspossible.

As a result of this screening, for example, 20 positive signals areobtained from 2×10⁵ plaques and further screening enables to isolatepositive clones. However, the size of a plaque, obtained on the basis ofa phage vector inserted with a fragment containing the objectivepromoter, is so small that minor contamination of other plaques willlead to an exclusive formation of DNAs originating from contaminatingphages upon extraction of phage DNAs, as a result of preferentialproliferation of the contaminating phages with a more rapid multiplyingrate either in the plate lysate method or in a culture broth method.

In fact, these problems have not been expected at all until thescreening is carried out. Thus, a plaque corresponding to the signal canbe obtained by carrying out repeated secondary screening, where adiluted solution of a primary phage is thinly sprayed, or by furthercarrying out tertiary screening. Surprisingly in the tertiary screening,a careful examination of the plate can detect a plaque, which is muchsmaller than other negative plaques, at a position corresponding to thesignal that can not be identified at first glance. The thus-obtained,very small plaque is handled so carefully as to prevent contamination ofother plaques in order to proliferate only the phage originating fromthe plaque. Extraction of a DNA fragment inserted in the phage vectorenable to afford, for example, a DNA fragment of an about 11 kbp length.

In addition, a clone containing the objective EXT gene can beeffectively screened in the secondary screening by carrying outconcurrently hybridization using an oligonucleotide VAN-U7 (SEQ ID NO17), synthesized on the basis of a 5'-uncoded region of a lower homologywith cDNAs of family genes other than EXT gene, as a probe.

Digestion of the DNA fragment with, for example, restriction enzyme HindIII (TAKARA SHUZO Co., Ltd.), followed by genomic Southern hybridizationusing the above-mentioned genomic DNA of EXT gene from azuki bean as aprobe, enables to define a shorter DNA fragment containing a promoterregion of this gene. This fragment is inserted into a restriction siteof a plasmid and the plasmid inserted with the fragment can betransferred into an appropriate host. Also, DNA sequencing analysisusing the plasmid inserted with said DNA fragment, followed bycomparison of the DNA fragment with the cDNA sequence of EXT gene,enables to judge whether the DNA fragment contains a promoter of EXTgene. Furthermore, the fragment can be used for subcloning as a fragmentcontaining the objective promoter-upstream region. The thus-obtainedsubcloned DNA fragment having Hind III and EcoR I at both termini isintegrated into the Hind III and EcoR I sites of pUC118 (TAKARA SHUZOCo., Ltd.) to allow to determine the nucleotide sequence. FIG. 1 showsthe restriction map of the fragment. This nucleotide sequence is shownin SEQ ID NO 20 in the Sequence Listing.

The plasmid integrated with the fragment containing the objectivepromoter-upstream region into the Hind III and EcoR I sites of pUC118 isdenoted as pVXP101, whereas E. coli JM109 strain transformed withpVXP101 is denoted and indicated as Escherichia coli JM109/pVXP101 andhas been deposited on Feb. 23, 1995 (the date of original deposit) atNational Institute of Bioscience and Human-Technology, Agency ofIndustrial Science and Technology, Ministry of Industrial Trade andIndustry (1-3, Higashi 1-Chome, Tsukuba-Shi, Ibaragi-ken, 305, Japan)under the accession No. FERM BP-5389, in accordance with the BudapestTreaty.

The above-mentioned procedures are not always applicable to cloning of aDNA fragment containing the objective promoter region. For example, thisis the case when the fragment exerts a fatal or growth-regressing actionagainst a host bacterium. In fact, the above-mentioned phage containinga promoter of EXT gene forms a plaque that is so small to be searchedand applied for the screening.

Then, PCR method is conceivable as an alternative method for cloning aDNA fragment containing a promoter region of the EXT gene.

PCR method to amplify such an unknown sequence involves inverse PCR [ThePlant Journal, 7, 157-164 (1995)] and PCR using a cassette [TANPAKUSHITUKAKUSAN KOUSO (Proteins, Nucleic Acids, and enzymes), 35, 3157-3163(1990)].

However, conventional inverse PCR is effective only for amplification ofDNA chains up to about 1 kbp in length, when a genomic DNA of a higheranimal or plant is used as a template. Also, PCR using a cassette issimilarly effective only for amplification of fragments up to about 1kbp in length.

In addition, the selection of a restriction enzyme for self ligation islimited in inverse PCR, wherein the use of a restriction enzymerecognizing 4 bp is vital for obtaining an amplified fragment. In PCRusing a cassette, it is required to test many cassettes having a varietyof restriction enzyme sites recognizing 6 bp, thereby requiring a lot oflabor. Moreover, there is a low probability that even a restrictionenzyme site recognizing 4 bp, not to mention a restriction enzyme siterecognizing 6 bp, exists for a DNA fragment containing an AT-rich,biased sequence like a promoter region, whereby amplification of a longtarget DNA is required both in inverse PCR and in PCR using a cassette.Then, the present inventors have found out that a conventional methodcapable of amplifying only short DNA chains can be improved so as to becapable of effectively amplifying DNAs larger than about 2 kbp in lengthby optimizing the self-ligation conditions and the use of TAKARA LA PCRKit (TAKARA SHUZO Co., Ltd.) in inverse PCR.

In addition, the present inventors have found out that two-stage PCRprocedures, wherein the reaction solution in a first PCR shall bediluted for the use as a template in a second PCR in order to amplifythe objective DNA fragment effectively, thereby enabling to solve theabove-mentioned problems.

For example, genomic DNAs prepared from the leaves of azuki bean aresubjected to complete digestion using restriction enzyme Hind III,followed by self-ligation with T4 DNA ligase (TAKARA SHUZO Co., Ltd.).Since the efficiency of the self-ligation reaction is greatly dependenton the volume of this reaction system, it is preferred to adjust thevolume of reaction system so as to make the DNA concentration less than4 μg/ml.

PCR is carried out using the thus-obtained cyclic genomic DNA as atemplate. Examples of a primer to be employed include sequences such asprimer VAN-UH1 (SEQ ID NO 21), primer VAN-L (SEQ ID NO 22), primerVAN-UH2 (SEQ ID NO 23), primer VAN-L16 (SEQ ID NO 24), primer VAN-UH3(SEQ ID NO. 25), and primer VAN-L3 (SEQ ID NO 26), which are synthesizedon the basis of sequences (SEQ ID NO 18 and SEQ ID NO 19) of theabove-mentioned EXT gene genome DNA of pVXG303.

The two-stage PCR procedures are effective in order to amplify theobjective DNA fragment efficiently. For example, a DNA fragment of about1.8 kbp can be amplified by carrying out the first PCR using theabove-mentioned primer VAN-UH1 (SEQ ID NO 21) and primer VAN-L (SEQ IDNO 22) as primers and then utilizing the resulting reaction product as atemplate for the second PCR using primer VAN-UH2 (SEQ ID NO 23) andprimer VAN-L3 (SEQ ID NO 26).

However, the amplification is not efficient, if the first PCR is carriedout in the same manner as described above and then the second PCR iscarried out using primer VAN-UH2 (SEQ ID NO 23) and primer VAN-L16 (SEQID NO 24) or primer VAN-UH3 (SEQ ID NO 25) and primer VAN-L3 (SEQ ID NO26) as primers. In other words, the amplification efficiency isdependent on the selection of primers. Accordingly, it is preferred tofind out the most suitable combination of primers from severalcombinations made.

The PCR reactions shall be carried out by following the protocol ofTAKARA LA PCR Kit (TAKARA SHUZO Co., Ltd.), except for the reactiontemperature and the cycle conditions. Thus, the first PCR is carried outusing 50 μl of the reaction solution at 94° C. (0.5 minute), 55° C. (1.0minute), and 72° C. (2 minutes) with 30 cycles and then the second PCRis carried out under the same conditions. Hereupon, it is desirable toprepare several diluted solutions of the first PCR reaction solution inorder to find out the best amount to be added in the second PCR.

The amplified product of about 1.8 kbp can be subcloned into, forexample, the Hinc II site of pUC119 (TAKARA SHUZO Co., Ltd.). Comparisonof nucleotide sequences at both termini of the fragment with thesequences (SEQ ID NO 18 and SEQ ID NO 19) of the above-mentioned. EXTgene genomic DNA, which previously are partially cloned, reveals whethersaid fragment is a DNA fragment containing a promoter of EXT gene thatis continued from the previous sequences. FIG. 2 shows the restrictionmap of the fragment. The nucleotide sequence of the fragment is shown inSEQ ID NO 27 in the Sequence Listing. The plasmid integrated with thisPCR product is denoted as pVXP-H3, whereas E. coli JM109 straintransformed with pVXP-H3 is denoted and indicated as Escherichia coliJM109/pVXP-H3 and has been deposited on February 17, 1995 (the date oforiginal deposit) at National Institute of Bioscience andHuman-Technology, Agency of Industrial Science and Technology, Ministryof Industrial Trade and Industry (1-3, Higashi 1-Chome, Tsukuba-Shi,Ibaragi-ken, 305, Japan) as the accession FERM BP-5388, in accordancewith the Budapest Treaty.

SEQ ID NO and SEQ ID NO 2 in the Sequence Listing show nucleotidesequences upstream from a gene encoding the N-terminal amino acidsequence of EXT that are composed of SEQ ID NO 18 and SEQ ID NO 19together with SEQ ID NO 27 in the Sequence Listing.

As a result of nucleotide sequence analysis and comparison of SEQ ID NO1 with SEQ ID NO 2 in the Sequence Listing, it is revealed that bothsequences are entirely the same except for only two differences in allregions downstream from the 782th residue A of SEQ ID NO 1 anddownstream from the residue A of SEQ ID NO 2. These two differencesinvolve a difference in the number of continuing A residues downstreamfrom the 829th residue A of SEQ ID NO 1 and downstream from the 921stresidue A of SEQ ID NO 2 (16 bases in SEQ ID NO 1 and 14 bases in SEQ IDNO 2) and a difference between the 947th residue T of SEQ ID NO 1 andthe 1037th residue C of SEQ ID NO 2. This observation reveals that thiscommon region possesses a region regulating the specific expression atthe site and stage required for the reconstitution of plant cell wallxyloglucan.

A DNA fragment containing a promoter region of a family gene can becloned by solving the problems in the same manner as the method forcloning a DNA fragment containing a promoter region of the EXT gene.Furthermore, comparison of the resulting cloned fragment with some ofthese family genes that surely are expressed specifically at the siteand stage required for the reconstitution of plant cell wall xyloglucanenables to identify a region necessary for the tissue-specificexpression in plants. Also, a region necessary for an especially intenseexpression at the logarithmic growth phase in culture cells can beidentified in the same manner.

Measurement of Expression Site and Expression Stage--NorthernHybridization

In order to analyze the expression by a promoter, for example, theexpression site and the expression stage in azuki bean plants can bemeasured by northern hybridization using EXT gene cDNA of azuki bean asa probe. Also, for example, the expression stage in tobacco culturecells can be measured by northern hybridization using EXT gene cDNA oftobacco as a probe.

The EXT gene cDNA of azuki bean and the EXT gene cDNA of tobacco can becloned by methods described, for example, in EP-0562836 A1 (1993) and JP7-79778 A.

RNA extraction from the azuki bean plants and plant tissues such as thetobacco culture cells can be carried out, for example, by the guanidinethiocyanate method or the phenol-SDS method. The thus-extracted totalRNAs can be subjected to, for example, analysis by agarose gelelectrophoresis, followed by transference on a membrane and thenhybridization using this membrane. The total RNA level can be preparedin the same level, for example, by comparing the rRNA levels in theagarose gel electrophoresis, thereby enabling to correctly compare thelevels of the expressed EXT gene mRNA.

For example, using azuki bean plants grown for 40 days after seeding,total RNAs, which are extracted from stems, buds, and leaves by theguanidine thiocyanate method, are subjected to agarose gelelectrophoresis followed by northern hybridization using, as a probe, aDNA having a sequence specific to the EXT gene cDNA that is differentfrom other family gene cDNAs. In this case, the filter obtained afterthe hybridization is washed under such intensified conditions that theabove-mentioned probe having a sequence specific to the EXT gene cDNAcan be paired only with an mRNA of the target EXT gene without pairingwith other family gene mRNAs, thereby enabling the detection of only theexpression of the objective EXT gene. Such expression can also beconfirmed by the size of the mRNA. Comparison of the levels of the EXTgene mRNA in each plant tissue enables to reveal that the EXT gene mRNAis expressed in all sites and is intensely expressed, particularly instems where the reconstitution of plant cell wall xyloglucan is active.Furthermore, using azuki bean sprouts, the total RNA, which is extractedfrom every 1 cm-long cuts of epicotyl, is subjected to the analysis withagarose gel electrophoresis followed by northern hybridization using theEXT gene cDNA as a probe. In this way, it can be revealed that theexpression is most intense in the site where the epicotyl grows greatly,namely, in the site where the reconstitution of plant cell wallxyloglucan is active.

The expression site for each family gene can be clearly defined bynorthern hybridization using a probe specific to the respective familygenes, in the same manner as mentioned above.

Each of Tobacco BY2 culture cells [Fermentation Technology Today, p.689, Issued by NIHON HAKKOU KOUGAKUKAI (Japan Fermentation TechnologySociety) in 1972] cultivated for 1, 4, 6, 8, and 10 days is collected bysuction filtration, immediately subjected to rapid freezing using liquidnitrogen, and then kept at -80° C. until RNA extraction is operated. Thetotal RNA extracted from these cells by the phenol-SDS method can besubjected to the analysis by agarose gel electrophoresis followed bynorthern hybridization using a DNA having a sequence specific to thetobacco EXT gene cDNA as a probe. Comparison of the expression enablesto reveal that the expression occurs in any time and is particularlyintense at the logarithmic growth phase (4 days). It can be alsorevealed that, conversely, the tobacco XRP1 gene, a family gene, isexpressed intensely at the stationary phase and is not expressed sointensely at the logarithmic growth phase.

Identification of Expression Site and Expression Stage by RT-PCR

Moreover, RT (Reverse Transcriptase)-PCR method can be employed toanalyze simply the expression controlled by family gene promoters ofthese EXT genes.

For example, each of stems, buds, and leaves of azuki bean plants grownfor 40 days after seeding is separately collected, immediately subjectedto rapid freezing using liquid nitrogen, and then kept at -80° C. untilRNA extraction is operated. The total RNAs are extracted from thesetissues, for example, by the guanidine thiocyanate method. Each of thetotal RNAs can be used as a template for RT-PCR using TAKARA RNA PCR Kit(TAKARA SHUZO Co., Ltd.) to identify the expression site. In this case,the expression-site specificity controlled by each family gene promotercan be identified by using a sequence specific to the respective familygene as a primer.

For example, using azuki bean plants grown in the dark for 5 days afterseeding, every 1 cm-long sections of epicotyl are separately collected,immediately subjected to rapid freezing using liquid nitrogen, and thenkept at -80° C. until RNA extraction is operated. Each of the totalRNAs, which are extracted from these tissues, for example, by theguanidine thiocyanate method, can be used as a template for RT-PCR usingTAKARA RNA PCR Kit (TAKARA SHUZO Co., Ltd.) to identify the specificityof the expression site and stage in more details.

For example, each of Tobacco BY2 culture cells cultivated for 0, 1, 2,4, 6, 8, and 10 days is collected by suction filtration, immediatelysubjected to rapid freezing using liquid nitrogen, and then kept at -80°C. until RNA extraction is operated. Each of the total RNAs extractedfrom these cells by the phenol-SDS method can be used as a template forRT-PCR using TAKARA RNA PCR Kit (TAKARA SHUZO Co., Ltd.) to identify theexpression stage. In this case, the expression-stage specificitycontrolled by each family gene promoter can be identified by using asequence specific to each of the tobacco EXT gene and its family gene asa primer.

Gene Direct Transfer and GUS-Activity Measurement--Transient Assay

The full length or a portion of a sequence containing theabove-mentioned promoter is cut off and ligated to a variety of reportergenes to prepare chimeric genes. The promoter activity can be measuredby direct transfer of such a chimeric gene into plant cells.

A reporter gene means a gene that is ligated at a downstream from thepromoter region of the objective gene in order to examine the promoteractivity of the gene or the action of other cis-elements. A codingregion of an E. coli-origin enzyme gene is principally utilized as thereporter, since the cells to be transformed with the chimeric geneshould not have the same or similar enzymatic activity.

Examples of such reporter gene in the case of plants include genes ofGUS of the E. coli-origin, chloramphenicol acetyltransferase (CAT),β-galactosidase (lacZ), neomycin phosphotransferase (NPTII), luciferase,etc, of which GUS of the E. coli-origin recently has been well utilizedparticularly.

The GUS activity is assayed by using 4-methylunbelliferylglucuronide(4-MUG, WAKO Pure Chemicals Industries. Ltd.) as the substrate andmeasuring the specific fluorescence emitted from its product,4-methylumbelliferone (4-MU, nacalai tesque). The measurement of 4-MUcan be easily carried out, since it is highly stable and the backgroundfluorescence is low. In addition, when5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc, Molecular Probes) isused as the substrate, the product is an insoluble indigo-blue pigment,called indigotin, and thus by utilizing its property, a localization ofthe GUS activity in cells or tissues can be easily examined.

Comparison of the promoter activity can be made by using, for example,the cauliflower mosaic virus 35S promoter that is contained in pBI121(Clontech) and pBI221 (Clontech).

The transcription can be efficiently terminated by linkage of atranscription-termination sequence at a downstream from the reportergene. The transcription termination sequence may be originated from EXTgene or may be originated from another gene. In addition, thetranscription efficiency can be enhanced by linkage of a poly-A additionsequence at a downstream from the inserted sequence. The poly-A additionsequence may be originated from EXT gene or may be originated fromanother gene, exemplified by Agrobacterium octopine synthetase [The EMBOJournal, 3, 835-846 (1984)] and Agrobacterium nopaline synthetase [TheJournal of Molecular and Applied Genetics, 1, 561-573 (1982)]. Such achimeric gene cassette can be inserted into an appropriate vector andamplified in E. coli as a plasmid in order to transfer directly into aliving organism.

A method for introducing a vector containing this chimeric gene into aliving organism is exemplified by the microinjection method [Molecular &General Genetics, 202, 179-185 (1986)], the polyethylene glycol method(Nature, 296, 72-74 (1982)], the particle gun method [Nature, 327, 70-73(1987)], the protoplast fusion method with a cassette DNA or anRNA-containing small cells, cells, lysosome, etc. [Proceedings of theNational Academy of Sciences of the USA, 79, 1859-1863 (1982)], theelectroporation method [Proceedings of the National Academy of Sciencesof the USA, 82, 5824-5828 (1985)], and so on.

A transient transcriptional expression of the thus-transferred gene inthe cells for initial several days can be utilized for the transientassay to analyze an expression product in an extract of cells that arecultivated for 1 to 2 days after the transfer.

The plasmid vector that can be amplified in E. coli is exemplified bythe cauliflower mosaic virus 35S promoter, the E. coli-origin GUS gene,and pBI121 (Clontech) having a transcription termination sequencecassette originating from nopaline synthetase. In order to remove thecauliflower mosaic virus 35S promoter region in the plasmid, thisplasmid is subjected to digestion with restriction enzymes Hind III andXba I (TAKARA SHUZO Co., Ltd.), followed by agarose gel electrophoresisto cut off the objective fragment other than the 35S promoter region. ADNA fragment containing a promoter region of the EXT gene can betransferred into this purified site.

The thus-prepared DNA fragment containing a promoter region of EXT geneand a vector containing a chimeric gene of GUS gene can be transferredinto the tobacco BY2 culture cells by using, for example, theelectroporation method.

In order to transfer into the tobacco BY2 culture cells by using theelectroporation method, the tobacco BY2 culture cells can be convertedto cell wall-free protoplasts by treatment with, for example, an enzymesolution (pH: 5.5) containing 1% cellulase-ONOZUKA (Yakult Honsha Co.,Ltd.), 0.1% pectolyase Y23 (SEISHIN Corpolation), and 0.4 M mannitol at30° C. for 2 hours. A suspension of the obtained 2×10⁶ protoplasts ofthe tobacco BY2 culture cells in an electroporation buffer solution (70mM KC1, 1.5 mM MES, and 0.3 M mannitol) is mixed with 3 pmol of a vectorDNA and a 10% PEG6000/electroporation buffer solution. An electric pulse(300 V, 125 μF) using, for example, Gene Pulser II (Bio-RadLaboratories) is applied to the resulting mixture to transfer the DNAinto the plant cells. The cells are then incubated in theLinsmaier-Skoog culture medium (Physiologia Plantarum, 18, 100 (1965)]containing 0.2 mg/l 2,4-D as an auxin, 1% sucrose, and 0.4 M mannitol at26° C. for 1 to 2 days after the transfer. The cells are extracted andGUS, the expression product, in the extract can be measured by thefluorescence analysis. That is to say, a mixture of the recovered cellsin 200 μl of an extraction buffer solution [50 mM phosphate buffer (pH7.0), 10 mM EDTA, 0.1% Triton X-100, 0.1% Sarkosyl, and 10 mM2-mercaptoethanol] placed in an Eppendorf tube is subjected toultra-sonication and a supernatant isolated by centrifugation is usedfor the assay of the GUS activity and the assay of the protein quantityto determine the GUS specific activity.

The GUS activity is assayed by measuring a specific fluorescence(excitation wavelength: 365 nm; fluorescence wavelength: 455 nm), forexample, emitted by 4-MU, the product, when 4-MUG is used as thesubstrate. That is to say, 45 μl of the extraction buffer solution and25 μl of 4 mM 4-MUG are added to react with each 30 μl of the extractplaced in a 96-well microtiter plate. After 5, 35, and 95 minutes, thereaction is terminated by addition of 50 μl of a reaction-terminationsolution (1 M Na₂ CO₃). Then, the specific fluorescence (excitationwavelength: 365 nm; fluorescence wavelength: 455 nm) emitted by 4-MU ismeasured with a fluorescence plate reader to assay 4-XU, the product,when 4-MUG is used as the substrate.

Moreover, the protein quantity is assayed by a procedure exemplified asfollows. Thus, 2, 5, 10, 15, 20, and 30 μl of a 1/5-diluted solution ofthe extract or an 800 μg/ml BSA standard solution (20 μl of the extractbuffer solution is mixed with 80 μl of 1 mg/ml BSA) are placed in a96-well microtiter plate and thereto are added respectively 158, 155,150, 145, 140, and 130 μl of distilled water and 40 μl of the assayreagent in Bio-Rad Protein Assay Kit (Bio-Rad Laboratories) to make atotal volume to 200 μl, each. After being stirred slowly and thenallowed to stand for 20 minutes at room temperature, the mixture ismeasured by a plate reader (wavelength: 590 nm) within 60 minutes toassay the protein quantity. At the same time when the above assays arecarried out, the fluorescence intensities of the 4-MU standard solutionsare measured and the results are plotted on a graph with the 4-MUquantity (pmol) at the x-axis and the fluorescence intensity at they-axis. The 4-MU quantity per one fluorescence unit is obtained from theslope. Furthermore, the results on the samples are plotted on a graphwith the time (minute) at the x-axis and the fluorescence intensity atthe y-axis to obtain the increasing rate of the fluorescence intensityand then to obtain the decomposition rate of 4-MUG equal to the GUSactivity. In addition, the GUS specific activity can be obtained fromthe amount of protein.

In this way, it can be confirmed that the DNA fragment containing theEXT gene promoter region exhibits an activity more intense than that ofthe cauliflower mosaic virus 35S promoter that has been said to beexpressed intensely in the plants.

Transformed Plants

The thus-obtained DNA fragment containing the EXIT gene promoter regionand the vector containing the inserted chimeric gene of the GUS gene canbe transferred into plants or plant cells to prepare transformants.

The vector into which a chimeric gene is inserted is preferred tocontain a selective marker gene so as to facilitate selection oftransformed plants or plant cells. For example, a gene providing anantibiotic resistant property (antibiotic-resistant gene) can beutilized as the selective marker gene. Such a gene can be exemplified bygenes providing a resistance against G418, hygromycin, bleomycin,kanamycin, gentamicin, and chloramphenicol. In the case where anantibiotic-resistant gene is integrated into a vector, the transformedplants or plant cells, namely, the plant or plant cells into which sucha cassette is transferred, can be easily selected by picking up plantsor plant cells that grow in a culture containing the antibiotic.

A method for introducing a vector containing the inserted chimeric genedirectly into plants is exemplified by the microinjection method, thepolyethylene glycol method, the particle gun method, the protoplastfusion method with a vector-containing small cells, cells, lysosome,etc., the electroporation method, and so on.

Moreover, a chimeric gene can be transferred into plants by utilizing aplant virus as a vector. For example, a cauliflower mosaic virus can beutilized as the plant virus. That is to say, a virus genome is firstinserted in a vector of the E. coli origin to prepare a recombinant andthen such a cassette is inserted into the virus genome. This cassettecan be inserted into plants by cutting out the thus-modified virusgenome from said recombinant using restrictive enzymes and then byinoculating the genome into plants (Molecular Biology of Plant Tumors,pp. 549-560, Issued by Academic Press in 1932 and U.S. Pat. No.4,407,956).

Furthermore, such a cassette can be transferred into plants by employingsuch a property of a bacterium of the Agrobacterium genus that, oninfection to a plant, a portion of its plasmid DNA is transferred into aplant. genome.

Of bacteria of the Agrobacterium genus, Agrobacterium tumefaciensinfects a plant to induce crown galls and also Agrobacterium rhizogenesinfects a plant to induce hairy roots, which are caused by the transferof a region called as the transferred DNA region in a bacterial plasmidcalled as the Ti plasmid or the Ri plasmid into the plant to beintegrated into the plant genome, when the bacteria infect the plant. Inaddition, there is another region called as the vir-region in the Tiplasmid or the Ri plasmid, which is essential for the T-DNA region to betransferred into the plant and then integrated into the plant. Thevir-region itself is not transferred into the plant and also thisvir-region can function on a plasmid other than that containing theT-DNA region [Nature, 303, 179-189 (1983)].

If the objective DNA to be integrated in the plant genome has beeninserted into the T-DNA region on the Ti plasmid or the Ri plasmid, theobjective DNA can be integrated into the plant genome when the bacteriaof the Agrobacterium genus infect the plant. Then, the portion causingcrown galls or hairy roots in the T-DNA region on the Ti plasmid or theRi plasmid is removed without spoiling the objective transferringfunction and the resulting plasmid can be utilized as a vector. Avariety of such vectors can be utilized in the present invention. Forexample, using pBI121 (Clontech) called as a binary vector, a GUS genesite linked to the cauliflower mosaic virus 35S promoter in pBI121 isreplaced by the DNA fragment containing the EXT gene promoter region andthe chimeric gene with the GUS gene to utilize for the transfer of saidchimeric gene into the plant. In this case, a simultaneous usage of avector having a promoter-free GUS gene (pBI101, Clontech) as a negativecontrol, pBI121 (Clontech), etc. enables to compare with the expressionmode of the cauliflower mosaic virus 35S promoter. Since such a vectordoes not have the vir-region, the bacteria of the Agrobacterium genusare required to contain another plasmid having the vir-region.

Moreover, this vector can be amplified not only in the bacteria of theAgrobacterium genus but also in E. coli. Accordingly, the recombinantoperation of the Ti plasmid can be carried out using E. coli. Inaddition, this vector includes an antibiotic-resistant gene and thus thetransformant can be easily selected, when E. coli, the bacteria of theAgrobacterium genus, and plants are transformed.

The transformation is applicable to any species of plants, provided thatthe plant can be infected by the bacteria of the Agrobacterium genus andestablishes the regeneration system. Most of dicotyledonous plants canundergo transformation using the bacteria of the Agrobacterium genusand, particularly, all plants that are hosts of bacteria of theAgrobacterium genus in the nature can be transformed in vitro. Althoughmonocotyledonous plants including cereals are not hosts of bacteria ofthe Agrobacterium genus in the nature, for example, rye plants [Nature,325, 274-276 (1987)], maize plants [Science, 240, 204-207 (1988)], riceplants [Nature, 338, 274-276 (1989)], and so on can be transformed invitro.

The transformation can be carried out (1) by using protoplasts and (2)by using a piece of tissues or untreated cells. For using method (1), itis required to establish in advance a system to regenerate the plantfrom transformed protoplasts. For using method (2), it is required totransform a piece of tissues or untreated cells by using the bacteria ofthe Agrobacterium genus and then establish a system to regenerate themin the plant. The transformed plant can be selected by growing in aculture medium containing an agent which can be the above-mentionedtransformation marker.

The method for regenerating the plant from the plant cells, albeitdifferent in the plant species, generally comprises deriving callus froma suspension of the transformed protoplasts in the case of (1) or fromthe piece of tissues or untreated cells that were transformed on theplate in the case of (2) and then forming shoots. In addition, theculture medium to be used for the regeneration may contain hormones suchas auxin or cytokinin in addition to a variety of amino acids.

Whether the objective cassette is inserted into the genome of thetransformed plant can be confirmed by Southern hybridization or thelike, whereas whether the reporter gene mRNA is formed in the plant canbe confirmed by northern hybridization or the like.

Utilizing the plant in which the chimeric gene prepared as describedabove is inserted, the chimeric gene can be transferred to the nextgeneration of plants by mating.

For example, a plasmid containing the DNA fragment containing the EXTgene promoter region and the chimeric gene with the GUS gene to beobtained by the present invention can be constructed in pBI121(Clontech). Next, the thus-constructed plasmid can be utilized fortransformation of an appropriate strain of the Agrobacterium genus suchas Agrobacterium tumefaciens LBA4404 strain [Nature, 303, 179-180(1983); available from Clontech], followed by infection of thetransformant to the objective plant to transform the plant.

For example, Arabidopsis seeds (available from Notlingham ArabidopsisStock Center: NASC) are aseptically cultivated on an MSO plate[MURASHIGE-Skoog inorganic salt mixture (WAKO Pure ChemicalsIndustries., Ltd.), mixed with 2% sucrose, 3 mg/l thiaminehydrochloride, 5 mg/l nicotinic acid, and 0.5 mg/l pyridoxinehydrochloride, is adjusted to pH 6.3, mixed further with 0.2% gellangum, autoclaved, and plated] according to a conventional procedure andthen cut sections of the roots are used for callus culture on the CIMplate (0.5 mg/l 2,4-dichlorophenoxyacetic acid and 0.05 mg/l kinetin areadded to the MSO plate).

Each of the Agrobacterium transformed by a plasmid containing theaforementioned chimeric gene and the Agrobacterium transformed by pBI121and pBI101 is cultivated and the diluted mixture is distributed intotubes. Then, sections of roots that callus formed are soaked therein andco-cultivated on a CIM plate for several days. When each bacterialstrain sufficiently grows to visible, they are killed by a bacteriaspecific antibiotic and sections of roots are cultivated further forseveral days on a SIMC plate [to the MSO plate are added 2-ip[N6-(2-isopentenyl)adenine (WAKO Pure Chemicals Industries, Ltd.)] at afinal concentration of 5 μg/ml, IAA (3-indoleacetic acid, WAKO PureChemicals Industries, Ltd.) at a final concentration of 0.15 μg/ml, andClaforan (Hoechst) at a final concentration of 500 μg/ml]. The resultingsections are finally cultivated on the SIMCS plate (the SIMC platecontaining kanamycin) and repeatedly transplanted on a new plate everyweek. The transformed cut sections keep growing to form swollen mass,whereas untransformed sections turn brown. The transformant iscultivated till formation of rosette leaves and the bottom of thetransformant is cut off with a scalpel so as not to contain the calluspart and transferred to a RIM plate (IAA is added at a finalconcentration of 0.5 μg/ml to the MSO plate). After 8 to 10 days, thecultivation is continued on a rock-fiber mini-box (NITTO BOUSEKI Co.,Ltd.) soaked in an inorganic salt culture medium [Hyponecks (HyponecksJapan)] is diluted 1000-fold with water]. After flowering and podding,the plant is transplanted in the soil soaked with the inorganic saltculture medium to obtain seeds. The seeds are sterilized and then sownand germinated on an MSK plate (kanamycin is added at a finalconcentration of 500 mg/l to the MSO plate) to obtain a transformant.

Whether or not the transformation occurs can be identified by extractionof a DNA from this transformant by a conventional method, cleavage ofthe DNA with restriction enzymes Hind III and EcoR I, and Southernhybridization using, as a probe, a promoter region that is prepared bydigestion of pVXP-H3 with restriction enzymes Hind III and EcoR I. Thatis to say, when the above procedure is carried out on (1) the WS strainthat does not undergo the transformation, (2) the transformant in whichthe chimeric gene is transferred, and (3) the transformant in which onlythe vector pBI121 is transferred, a specific signal of about 1.8 kbp,besides endogenous signals common in (1) to (3), is observed in a sampleof (2) digested with restriction enzymes Hind III and EcoR I, therebyidentifying that the DNA containing the EXT gene promoter is integratedinto (2).

In the case where the thus-obtained transformant is assayed for the GUSactivity using X-Gluc as the substrate, localization of the GUS activityin cells or tissues can be easily examined by utilizing the property ofthe product called as indigotin, an insoluble indigo-blue pigment. Thatis to say, young plants, obtained by sowing the sterilized seeds on theMSK plate (kanamycin is added at a final concentration of 50 mg/l to theMSO plate) followed by germination, are placed, as is, in watercontaining 2 mM DTT and, after deaeration under reduced pressure, aretransferred, as is, into the GUS reaction solution [1 mM X-Gluc, 50 mMphosphate buffer solution (pH 7.0), and 20% methanol] to undergo thereaction at 37° C. for 0.5 to 4 hours.

After completion of the reaction, ethanol is added to stop the reactionand remove pigments such as chlorophyll and the plants were washed withethanol two or three times, allowed to stand for 3 hours to overnight,and then transferred in a Petri dish filled with water. The plants areplaced on a slide glass and, after addition of 1 to 2 drops of 70%hydrous glycerol for fitting followed by further addition of glycerol,pressed with a cover glass to allow to be observed under a microscope.When the above procedure is carried out on (1) the WS strain that doesnot undergo the transformation, (2) the transformant in which thechimeric gene is transferred, and (3) the transformant in which only thevector pBI121 is transferred, it can be observed that tissue portionswhich grow with elongation in (2) are stained blue, whereas tissues in(1) are not stained at all and the whole tissues in (3) are stained,albeit unevenly.

The procedure exemplified in the following can be applied to a procedurefor obtaining a promoter that is hybridizable to the plant promoter ofthe present invention and also possesses the promoter activity in atleast one of plants, plant cells, and transgenic plants regenerated fromthe plant cells.

First, a chromosomal DNA obtained from the objective gene source istransferred into a host by ligation to a plasmid or phage vector,according to a conventional method, to prepare a library. The library iscultivated on a plate and grown colonies or plaques are taken on anitrocellulose or nylon membrane, on which the DNA is immobilized bydenaturation. The membrane is warmed in a solution containing a probethat is labeled in advance with ³² P etc. (the probe is exemplified bythe nucleotide sequences as described in SEQ ID NO 1, 2, 3, 4, 5, 6, 7,and 8 in the Sequence Listing or some genes thereof) to hybridize theDNA on the membrane with the probe. For example, the DNA-immobilizedmembrane undergoes hybridization with the probe at 65° C. for 20 hoursin a solution containing 6×SSC, 1% sodium lauryl sulfate, 100 μg/ml of asermon sperm DNA, and 5×Denhardt's solution (containing bovine serumalbumin, polyvinyl pyrrolidone, and ficoll, each at a 1% concentration).After the hybridization, nonspecific adsorption is washed away and aclone hybridized with the probe is identified by autoradiography or thelike. This process is repeated until a single hybridized clone isobtained. The objective plant promoter is inserted into thethus-obtained clone.

The nucleotide sequence of the resulting gene is determined, forexample, by the following way to confirm that this gene is the objectiveplant promoter.

In the case of nucleotide sequencing of the clone obtained by thehybridization, E. coli, when used as the recombinant, is cultivated intest tubes or the like and a plasmid is extracted by a conventionalmethod. After the cleavage with restriction enzymes, the insertedfragment is taken out and undergoes subcloning to the M13 phage vector,followed by the nucleotide sequencing by the dideoxy method. In the casewhere the recombinant is a phage, the nucleotide sequencing can becarried out by basically the same steps. Basic experimental proceduresfor such cultivation to nucleotide sequencing are described in"Molecular Cloning, A Laboratory Manual" (T. Maniatis et al., publishedby Cold Spring Harbor Laboratory Press in 1989) and so on.

In order to confirm that the obtained gene is the objective plantpromoter, the determined nucleotide sequence is compared with that ofthe plant promoter of the present invention and with the nucleotidesequences as described in SEQ ID NO 1, 2, 3, 4, 5, 6, 7, and 8 in theSequence Listing to deduce the structure of the gene.

When the obtained gene does not contain the entire plant promoterregion, a synthetic DNA primer is prepared on the basis of the obtainedgene and then the nucleotide sequence of the entire plant promoterregion that hybridizes the promoter of the present invention can bedetermined by amplification of a deficient region by PCR and furtherscreening of the DNA library using the obtained gene fragment as aprobe.

Moreover, on the basis of the nucleotide sequence of the plant promoterof the present invention, modification of a portion of the nucleotidesequence by at least one of substitution, insertion, and deletion thatare induced by site-directed mutagenesis of the gene containing thenucleotide sequence enables to change the function of the plant promoterof the present invention, thereby obtaining plant promoters similar tothe plant promoters of the present invention. Examples of known methodsfor such site-directed mutagenesis include the gapped duplex method[Methods in Enzymology, 154, 350-367 (1987)], the uracil-containing DNAmethod [Methods in Enzymology, 154, 367-382 (1987)], the nitrite method[Proceedings of the National Academy of Sciences of the USA, 79,7258-7262 (1982)], and further the cassette mutagenesis method [Gene,34, 315-323 (1985)].

Furthermore, on the basis of the nucleotide sequence of the plantpromoter of the present invention, a chimeric promoter [Proceedings ofthe National Academy of Sciences of the USA, 88, 7266-7270 (1991)] isconstructed by ligation or substitution of a gene containing thenucleotide sequence or a portion of the gene with a gene of known plantpromoters or the like, or a portion of this gene, thereby obtaining aplant promoter possessing the promoter activity at the site and stagerequired for the reconstitution of plant cell wall xyloglucan, like thepromoters of the present invention.

Ligation of the thus-obtained plant promoter at a downstream therefromwith a useful gene in the operable state followed by assay of thepromoter activity by the same method as that for the promoters of thepresent invention enables to confirm whether the plant promoterfunctions in at least one of plants, plant cells, and transgenic plantsregenerated from the plant cells. Also, the expression site specificitycontrolled by the plant promoter can be identified.

In the case where the plant promoter to be obtained in the presentinvention is introduced into any of plants, plant cells, and transgenicplants regenerated from the plant cells, the promoter can be introducedin the form of a vector that is retained extrachromosomally or of avector that is integrated intrachromosomally. Such extrachromosomallyretained vectors and intrachromosomally integrated vectors are known inthe art and introduction into plants and transgenic plants regeneratedfrom plant cells can be carried out by, for example, the microinjectionmethod, the polyethylene glycol method, the particle gun method, theprotoplast fusion method with a vector-containing small cells, cells,lysosome, etc., the electroporation method, and so on. Furthermore, thevector can be converted into a chimeric gene in which a gene encodingseveral amino acids at the N-terminus of an enzyme possessing a functionto reconstitute plant cell wall xyloglucan at a downstream from theplant promoter is ligated at an upstream from the useful gene in anoperable state.

Using promoters of the EXT gene or EXT family genes to be obtained inthe present invention or polynucleotides having a sequence or itspartial sequence that are hybridizable to the promoters, ligation of auseful gene at its downstream in an operable state, followed byintroduction into plants and plant cells, enables to induce the geneexpression in a specific manner at the site and stage required for thereconstitution of plant cell wall xyloglucan, like the promoters of thepresent invention, thereby controlling the plant morphology. Forexample, the control can be made by ligating a gene encoding anantisense RNA, a decoy etc. or a ribozyme so as to function at adownstream from the promoter of the present invention, followed byintroduction into plants, plant cells, and transgenic plants regeneratedfrom the plant cells. Dwarf plants can be produced by controlling theplant morphology, whereas male-sterile plants can be prepared bycontrolling elongation of the pollen tube. Furthermore, the quality offood or feed can be improved for plants of which the elongating/growingstems, the sites required for the reconstitution of plant cell wallxyloglucan, are utilized as foods. Further, induction of specific geneexpression at the logarithmic growth phase or the stationary state ofculture cells enables, for example, control of the cell proliferationand improvement in the productivity of useful secondary metabolites.

Moreover, according to the present invention, plant promoters having thepromoter activity at the site and stage required for the reconstitutionof plant cell wall xyloglucan can be cloned by utilizing the genesencoding enzymes having the function to carry out the reconstitution ofplant cell wall xyloglucan and, particularly, genes encoding EXT or itsfunctional equivalent.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a restriction map of the fragment inserted in pVXP101.

FIG. 2 is a restriction map of the fragment inserted in pVXP-H3.

FIG. 3 is a photograph illustrating a migration pattern of northernhybridization of the culture cells in Example 10.

FIG. 4 is a graph illustrating the growth of the culture cells inExample 10.

FIG. 5 is a graph illustrating the results of transient assay in Example11.

FIG. 6 is a construction diagram of pBVEG101.

FIG. 7 is a construction diagram of pBVEG121.

FIG. 8 is a construction diagram of pBI-H-101.

FIG. 9 is a construction diagram of pBI-H-121.

FIG. 10 is a photograph illustrating the results of GUS-staining of thetransgenic Arabidopsis plant in Example 12.

FIG. 11 is a restriction map of the DNA fragment of about 6.0-kbpamplified by PCR in Example 13.

FIG. 12 is a restriction map of the DNA fragment of about 4.5-kbpamplified by PCR in Example 14.

FIG. 13 is a restriction map of the fragment inserted in pXRG302.

FIG. 14 is a restriction map of the fragment inserted in pLXG101.

FIG. 15 is a restriction map of the fragment inserted in pNXG102.

FIG. 16 is a restriction map of the fragment inserted in pKOM-1.

FIG. 17 is a restriction map of pKEP-1.

FIG. 18 illustrates an electrophoresis migration patter of northernhybridization of the plant in Example 19.

FIG. 19 illustrates an electrophoresis migration patter after RT-PCR ofthe plant in Example 19.

FIG. 20 is a construction diagram of pVAEXT2GUS.

FIG. 21 is a construction diagram of pVAEXT3GUS.

FIG. 22 is a construction diagram of pVAXRP1tlGUS.

FIG. 23 is a construction diagram of pLEEXT1.4GUS.

FIG. 24 is a construction diagram of pLEEXT0.7GUS.

FIG. 25 is a construction diagram of pLEEXTtl4.9GUS.

FIG. 26 is a construction diagram of pLEEXTtl1.4GUS.

FIG. 27 is a construction diagram of pNTEXT0.8GUS.

FIG. 28 is a construction diagram of pTAEXT1.1GUS.

FIG. 29 is a graph illustrating the comparison of the GUS specificactivities of the transformed tobacco culture cells in Example 11 andExample 20.

The following examples further illustrate the present invention in moredetail but are not construed to limit the scope of the presentinvention.

EXAMPLE 1

Isolation of Family Genes (Azuki Bean EXT 2 and Azuki Bean EXT 3) ofEndo-xyloglucan Transferase (EXT)

(1) Poly(A)⁺ RNA

Seeds of Vigna angularis ohwi et Ohashi, cv. Takara (WATANABE SHUSHICo., Ltd.) were germinated according to the method described inPhysiologia Plantarum, 82, 490-497 (1991).

After one week from the germination, stems and leaves in the ground partwere cut off to obtain about 2 g of plant tissues. They were immediatelyfrozen in liquid nitrogen and grounded in a mortar in the presence ofliquid nitrogen to prepare a powder, which was suspended in 20 ml of adenaturation solution [7 M guanidine thiocyanate, 25 mM sodium citrate(pH: 7.0), 0.1 M mercaptoethanol, and 2% sodium lauroylsarcosinate). Theresulting suspension was crushed with Polytron, mixed with 10 ml of thedenaturation solution and 30 ml of a phenol/chloroform solution [a 1:1mixture of water saturated acidic phenol and chloroform/isoamyl alcohol(49:1)] with stirring thoroughly, and centrifuged to separate an aqueouslayer, which was mixed with isopropyl alcohol, a 1/10 volume of 3 Msodium acetate, and a 1/300 volume of acetic acid and then centrifugedto obtain about 4 mg of RNA as a precipitate.

This precipitate was dissolved in 2 ml of an adsorption buffer solution[20 mM Tris-HCl (pH: 7.5), 2 mM EDTA, 1 M NaCl, and 0.5% SDS) andadsorbed on an oligo(dT)-cellulose Type-7 column (Pharmacia), which waseluted with an elution buffer solution [10 mM Tris-HCl (pH: 7.5) and 1mM EDTA] to recover about 25 μg of poly(A)⁺ RNA.

(2) Construction and Screening of cDNA Library

A cDNA library was constructed from poly(A)⁺ RNA obtained in Example1-(1) by using cDNA Synthesis Kit System-Plus (Amersham) according tothe method described in Gene, 25, 263-269 (1983) using λgt10(Stratagene) as a vector. The azuki bean EXT gene cDNA [EP-0562836 A1(1993)] was labeled with [α-³² P]dCTP using Random Primer Labeling Kit(TAKARA SHUZO Co., Ltd.) to prepare a probe for hybridization. Thespecific activity of this probe was 7.5×10⁸ cpm/μg. The plaquehybridization method using this probe was applied to theabove-constructed cDNP library. That is to say, plaques were formed at1×10⁴ plaques per plate and transferred on a membrane. Afterdenaturation, neutralization, and immobilization, the membrane wassubjected to pre-hybridization in a prehybridization buffer solution(6×SSC, 0.1% SDS, 5× Denhardt's solution, and 10 μg/ml salmon-sperm DNA)at 50° C. for 2 hours. Then, the hybridization buffer solution was addedto make the probe at 2×10⁵ to 10⁶ /ml and hybridization was carried outat 50° C. for 15 hours. After completion of the hybridization, themembrane was washed twice with a washing solution containing 6×SSC and0.1% SDS at room temperature for 20 minutes. The membrane was exposedovernight at -80° C. in a cassette in which a sensitized X-ray filmpaper (Kodak) was placed to prepare an autoradiograph. As a result ofsearch on 5×10⁴ plaques, 96 positive plaques were obtained. Each ofthese plaques underwent a secondary screening and used in the followingexperiment.

(3) Classification of Plaques and Isolation of Azuki Bean EXT2 cDNA andAzuki Bean EXT3 cDNA, Family Genes of EXT Gene

The plate lysate method (T. Maniatis et al., "Molecular Cloning, Alaboratory Manual", Second Edition, Chapter 2, pp. 60-66, published byCold Spring Harbox Laboratory Press in 1989) was applied to theabove-obtained plaques to prepare a large quantity of phage particles,which were employed for dot hybridization using, as a probe, the azukibean EXT gene cDNA used in Example 1-(2). After the hybridization stepscarried out in the same manner as described above, filters were washedunder gradiently intensified conditions with 6×SSC, 4×SSC, 2×SSC, and0.1×SSC at 50° C. and then 0.1×SSC at 65° C. to allow classification onthe basis of the signal intensities. As a result, the plaques wereclassified into 6 types of groups. Of these groups, two types of plaquesshowing signal intensities different from those of the azuki bean EXTgene were obtained from groups where the signals were detectable afterwashing with 0.1×SSC at 50° C. but were not detectable after washingwith 0.1×SSC at 65° C. Phages were isolated from these plaques and theinserted DNAs were extracted. The lengths of the DNA fragments wereidentified by cleavage of said DNAs with restriction enzyme EcoR Ifollowed by agarose gel electrophoresis. As the result, about 730 bp andabout 430 bp were detected from one type of plaque and about 1090 bpfrom another type of plaque. Each of these DNA fragments were subjectedto purification followed by subcloning at the EcoR I site of pUC18(TAKARA SHUZO Co., Ltd.) and the resulting plasmids were named aspVX-44-1, pvX-44-2, and pVX-45-1, respectively. When these plasmidsunderwent nucleotide sequencing of the DNA fragments according to theaforementioned Sanger method [Science, 214, 1205-1210 (1981)] usingBcaBEST™ Dideoxy Sequencing Kit (TAKARA SHUZO Co., Ltd.), a gene (azukibean EXT2) having a high homology with the azuki bean EXT gene wascloned from pVX-44-1 and pVX-44-2, and further another type of gene(azuki bean EXT3) having a high homology with the azuki bean EXT genewas cloned from pVX-45-1. Partial nucleotide sequences thereof are shownin SEQ ID NO 11 and SEQ ID NO 12 in the Sequence Listing.

EXAMPLE 2

Isolation of Azuki Bean XRP1 cDNA, Family Gene of EXT Gene

A cDNA library was constructed from poly(A)⁺ RNA obtained in Example1-(1) by using cDNA Synthesis Kit (TAKARA SHUZO Co., Ltd.) according tothe method described in Gene, 25, 263-269 (1983) using λZAPII(Stratagene) as a vector. In order to prepare a probe, a mixed syntheticoligonucleotide (27 mer, SEQ ID NO 13) corresponding to the amino acidsequence (SEQ ID NO 28) of DEIDFEFLG, one of sequences conserved oftenfor the proteins that act upon xyloglucans, was labeled with [γ-³² P]ATPusing DNA 5'-Terminal-Labeling Kit MEGALABEL™ (TAKARA SHUZO Co., Ltd.).The specific activity of this probe was about 1×10⁸ cpm/μg. The plaquehybridization method using this probe was carried out for theabove-constructed cDNA library in the same manner as in Example 1, wherethe hybridization was carried out at 50° C. for 15 hours. Then, themembrane was washed twice with 2×SSC at 50° C. for 20 minutes andunderwent the autoradiography. Plaques were formed on 10×14 cm plates atabout twenty thousand plaques per a plate. As a result of search onabout one hundred thousand plaques, no positive signals were detected.When a positive control was carried out at the same time by formation ofplaques of phage particles integrated with the azuki bean EXT cDNA atabout 50 plaques per a square plate, followed by hybridization under thesame conditions, distinct positive signals corresponding each of plaqueswere detected.

In contrast, it was strange that no positive signals were detected, whenthe phage solution (containing about ten thousand plaques) of the cDNAlibrary was mixed with the phage solution in the positive control so asto form about 50 plaques thereof per a square plate. Such an incidencehas not occurred in the case of plaque hybridization using a cDNAfragment with a length of more than 100 bases and thus the problemapparently arises when an oligomer of an about 20 to 30 base length isutilized as a probe. It is conceivable that the formation of positivesignals was blocked by a certain interaction with plaques originatingfrom a large number of other coexisting phages, even in the presence ofpositive clones.

As a result of the formation of plaques at 500 to 1000 plaques per aplate not so densely to avoid the problem, followed by search on about8×10³ plaques, 8 positive plaques were obtained. Upon double infectionwith the M13 helper phage and with the host bacterium of the F' strain,λZAPII can undergo automatic subcloning in which a region cloned in thehost is converted automatically into pBluescript SK (-) (Stratagene).Plasmids were prepared from the above-mentioned 8 plaques in this wayand were subjected to cleavage with the restriction enzyme EcoR I(TAKARA SHUZO Co., Ltd.) followed by agarose gel electrophoresis toidentify the lengths of DNA fragments. Of these fragments, oneoptionally selected DNA fragment of about 1.2 kbp was integrated into 3types of plasmids which were named as pVM104, pVM106, and pVM109,respectively.

When these plasmids underwent nucleotide sequencing of the DNA fragmentsaccording to the aforementioned Sanger method using BcaBEST™ DideoxySequencing Kit (TAKARA SHUZO Co., Ltd.), 2 types of genes having a highhomology with the azuki bean EXT gene as well as with the BRUL gene[Plant Physiology, 104, 161-170 (1994)] and the meri-5 gene [the PlantCell, 3, 359-370 (1991)] were cloned (pVM106 and pVM109 were theidentical gene). A partial nucleotide sequence of pVM106 (azuki beanXRP1), one of these genes, is shown in SEQ ID NO 14 in the SequenceListing.

EXAMPLE 3

Isolation of Azuki Bean XRP2 cDNA, Family Gene of EXT Gene, by PCR

About 10000 pfu (plaque forming unit) of a μphage solution (33 μl),prepared from the ground parts of azuki bean in the same manner as inExample 2, was subjected to twice extraction with the phenol/chloroformsolution followed by ethanol precipitation to obtain a simply purifiedμphage DNA. With this total DNA utilized as a template, the PCR reactionusing PCR Amplification Kit (TAKARA SHUZO Co., Ltd.) was carried out byusing the mixed synthetic oligonucleotide (Sequence ID NO 13) as a senseprimer and, as an antisense primer, an oligo(dT)18 primer in which dTTPwas bonded with 18 bases. The reaction was carried out at 94° C. (1minute), 55° C. (1 minute), and 72° C. (1 minute) with repeating thecycle 25 times and the resulting reaction solution was maintained at 72°C. for 7 minutes. Then, with 1 μl of this reaction solution utilized asa template, the second PCR was carried out by using the mixed syntheticoligonucleotide (SEQ ID NO 13) as a sense primer and the oligo(dT)18primer as an antisense primer, with repeating the above-mentioned cycle25 times. After completion of the reaction, the reaction mixture wasanalyzed by 3% agarose gel electrophoresis to confirm that DNA fragmentsof about 260 bp, 350 bp, 450 bp, 500 bp, 600 bp, 750 bp, 800 bp, and1300 bp were amplified in a specific manner. These fragments wererecovered and end-blunted by using DNA Blunting Kit (TAKARA SHUZO Co.,Ltd.). In addition, the 5' terminus of the PCR product wasphosphorylated by using MEGALABEL™ (TAKARA SHUZO Co., Ltd.) and theresulting product was subcloned at the Hinc II site of pUC119 (TAKARASHUZO Co., Ltd.). When these plasmids underwent nucleotide sequencing ofthe DNA fragments according to the Sanger method using BcaBEST™ DideoxySequencing Kit (TAKARA SHUZO Co., Ltd.), 2 types of genes having ahomology with the azuki bean EXT gene were cloned. One of them wasidentical with pVM106 and pVM109 in Example 2. A partial nucleotidesequence of another gene (azuki bean XRP2) is shown in SEQ ID NO 15 inthe Sequence Listing.

EXAMPLE 4

Isolation of Tobacco XRP1 cDNA, Family Gene of EXT Gene

A cDNA library (Stratagene) with λZAP as a vector was utilized and, inorder to prepare a probe, a mixed synthetic oligonucleotide (27 mer, SEQID NO 13) corresponding to the amino acid sequence (SEQ ID NO 28) ofDEIDFEFLG, conserved in the proteins that act upon xyloglucan, waslabeled with [γ-³² P]ATP using DNA 5'-Terminal-Labeling Kit MEGALABEL™(TAKARA SHUZO Co., Ltd.). The plaque hybridization using this probe wascarried out for the above-mentioned cDNA library in the same manner asin Example 2. Then, as a result of search on about 3×10⁴ plaques, 30positive plaques were obtained.

Upon double infection with the M13 helper phage and with the hostbacterium of the F' strain, λZAP (Stratagene) can undergo automaticsubcloning in which a region cloned in the host is convertedautomatically into pBluescript SK (-) (Stratagene). Plasmids wereprepared from the above-mentioned 30 plaques in this way and weresubjected to cleavage with restriction enzyme EcoR I followed by agarosegel electrophoresis to identify the lengths of DNA fragments. Of thesefragments, 2 types of plasmids containing DNA fragments of about 1.5 kpband about 0.9 kpb were named as pTM3D and pTM11D, respectively.

When these plasmids underwent nucleotide sequencing of the DNA fragmentsaccording to the Sanger method using BcaBEST™ Dideoxy Sequencing Kit(TAKARA SHUZO Co., Ltd.), 2 types of genes having a homology with theazuki bean EXT gene as well as the above-mentioned BRU1 gene and meri-5gene were cloned. A partial nucleotide sequence of pTM11D, one such type(tobacco XRP1), is shown in SEQ ID NO 16 in the Sequence Listing.

EXAMPLE 5

Isolation of Genome DNA Clones of EXT Gene Family Genes

(1) Preparation of Genome DNA from Azuki Bean Leaves

Seeds of Vigna angularis ohwi et Ohashi, cv. Takara (WATANABE SHUSHICo., Ltd.) were germinated according to the method described inPhysiologia Plantarum, 82, 490-497 (1991) to obtain about 10 g ofleaves. These leaves (about 10 g) were pulverized in a mortar in thepresence of liquid nitrogen to prepare a white powder. About 2.5 g ofthe leave powder was immediately placed in a 50 ml polystyrene tube andextracted with 10 ml of a urea-phenol DNA extraction buffer solution[0.05 M Tris-HCl (pH: 7.6), 0.02 M EDTA, 5% phenol, 8 M urea, 0.35 KNaCl, and 2% sodium lauroylsarcosinate] mixed with 25% SDS at 65° C. for1 hour. The extract was mixed with a 2-fold volume of aphenol-chloroform-isoamyl alcohol (25:24:1) mixture, stirred gently forabout 15 minutes, and then centrifuged at 2000 rpm for 15 minutes. Afterthe centrifugation, the supernatant was transferred into a new tube,again mixed with a 2-fold volume of a phenol-chloroform-isoamyl alcohol(25:24:1) mixture, stirred gently for about 15 minutes, and thencentrifuged at 2000 rpm for 15 minutes. The supernatant after thiscentrifugation was transferred into a new tube, mixed with a 2-foldvolume of ethanol, and stirred gently. Then, the precipitated, whitegenome DNA was coiled out by using a Pasteur pipet and transferred intoa new tube. To this tube was added 1.5 ml of a TE buffer solution (10 mMTris-HCl (pH: 8.0) and 1 mM EDTA] and the resulting mixture was kept at55° C. overnight to dissolve the DNA. Analysis of 1 μl of a sample,prepared by diluting of this DNA solution 10-fold, by 0.4% agarose gelelectrophoresis revealed that the solution contained a high molecularDNA at a concentration of about 100 ng/μl. In other words, 150 μg of thegenome DNA was obtained from about 2.5 g of the leaves.

(2) Construction of Genome DNA Library

Conditions were examined in order to subject the above-obtained genomeDNA to partial digestion with restriction enzyme Sau3A I. That is tosay, 10 U/μl of Sau3A I (TAKARA SHUZO Co., Ltd.) was diluted with adiluent buffer solution to adjust its concentration in a 50 μl reactionsolution (1 μg DNA) to 1 to 0.035 U/μg DNA, which was reacted at 37° C.for 30 minutes and then mixed with 1 μl of 0.5 M EDTA to stop thereaction. After the reaction, a 20 μl sample was analyzed by 0.4%agarose gel electrophoresis to indicate that molecules of 15 to 20 kbpsize were formed most abundantly under the condition with 0.1 U/μg DNA.The reaction was scaled up under this condition and to 10 μg of DNA,partially digested under this condition, were added 5 μl of a 10×fill-inbuffer solution [0.5 M Tris-HCl (pH: 7.2), 0.1 M magnesium sulfate, 1 mMDTT, 500 μg/ml acetylated BSA, 10 mM dATP, and 10 mM dGTP) and 10 U ofthe Klenow fragment. After the total volume was made 50 μl withdistilled water, the reaction was carried out at 37° C. for 30 minutes.After completion of the reaction, the reaction solution was mixed with50 μl of a phenol-chloroform-isoamyl alcohol (25:24:1) mixture, stirredgently, and then centrifuged at 12000 rpm for 5 minutes. The supernatantwas transferred into a new tube and precipitated with ethanol. Theprecipitate was dissolved in 20 μl of a TE buffer solution [10 mMTris-HCl (pH: 8.0) and 1 mM EDTA]. Then, 0.5 μg and 1.5 μg each of theresulting partially filled-in, partially Sau3A I-digested genome DNA wasligated with 1.0 μg of μGEM11 Arm (Promega Biotech) using TaKaRaLigation Kit Version 1 (TAKARA SHUZO Co., Ltd.). In other words, 0.5 μgand 1.5 μg each of the partially filled-in, partially Sau3A I-digestedgenome DNA was mixed with a solution containing 1.0 μg of λGEM11 Arm(Promega Biotech) and, after evaporation to dryness, the mixture wasdissolved in 5 μl of a ligation buffer solution, mixed with 5 μl ofSolution B in TaKaRa Ligation Kit Version 1 and then underwent ligationat 26° C. for 10 minutes. The ligated sample was subjected to twicephenol extraction followed by ethanol precipitation. Then, the totalamount underwent packaging by using an in vitro packaging kit(Stratagene), followed by infection with E. coli LE392, the hostbacterium, to obtain a genome DNA library of the azuki bean. The titerof this library was measured to be 2.1×10⁵ pfu/ml.

(3) Screening of Library and Isolation of Gene

Utilizing this library, the azuki bean EXT gene cDNA [EP-0562836 A1(1993)] of about 1.1 kbp was labeled with [α-³² P]dCTP using BcaBEST™Labeling Kit (TAKARA SHUZO Co., Ltd.) to prepare a probe for plaquehybridization. In other words, 25 ng of the above-mentioned DNA fragmentand 2 μl of a random primer were placed into a tube, diluted withdistilled water to make 5 μl, and subjected to heating at 95° C. for 3minutes followed by rapid cooling in ice. Thereto were added 2.5 μl of abuffer solution of a 10-fold concentration, 2.5 μl of a dNTP mixedsolution, 5 μl of labeled dCTP, distilled water to make 24 μl, and 1 μlof BcaBEST™ DNA Polymerase (TAKARA SHUZO Co., Ltd.) and the resultingsolution was incubated at 52° C. for 10 minutes. The enzyme wasdeactivated by heat denaturation with heating at 95° C. for 3 minutesfollowed by rapid cooling in ice. The total amount was used for thehybridization. The specific activity of this probe was 7.2×10⁸ cpm/μg.

The plaque hybridization was carried out in the same manner as inExample 1, except that the pre-hybridization and hybridization werecarried out at 65° C.

After the hybridization, the membrane was washed once with a washingsolution containing 2×SSC and 0.1% SDS at room temperature for 20minutes and further with a washing solution containing 0.1×SSC and 1%SDS at 50° C. for 20 minutes. Phages were inoculated on 10 square platesso as to form plaques at 1×10⁴ plaques per plate. As the result ofscreening on 1×10⁵ phages obtained from a total of 10 plates, 10positive plaques were obtained. Next, each of these plaques was utilizedfor secondary screening. Phage DNA was extracted from each plaqueobtained in the secondary screening by the plate lysate method. Thisphage DNA was subjected to digestion with restriction enzymes Sac I,EcoR I, Hind III, BamH I, and Pst I (all: TAKARA SHUZO Co., Ltd.),followed by Southern hybridization using the same probe mentioned above.

The Southern hybridization is carried out according to the methoddescribed in "Molecular Cloning, A laboratory Manual", Second Edition,Chapter 9, pp. 9.31-9.58 (T. Maniatis et al., Issued by Cold SpringHarbor Laboratory Press in 1989).

That is to say, each of the DNA samples was subjected to 1% agarose gelelectrophoresis, followed by alkaline denaturation and Southern blottingon a nylon membrane (Hybond-N, Amersham) overnight. After DNA wasimmobilized by irradiation with a ultraviolet transilluminator (254 nm)for 5 minutes, the membrane was subjected to pre-hybridization in 5 mlof a pre-hybridization buffer solution (5×Denhardt's solution, 6×SSC,0.1% SDS, and 10 μg/ml salmon sperm DNA) at 65° C. for 2 hours. Then,the probe was added and hybridization was carried out at 65° C.overnight. After the hybridization, the membrane was washed twice with awashing solution containing 2×SSC and 0.1% SDS at room temperature for10 minutes and then washed twice with the same washing solution at 50°C. for 30 minutes. After being dried, the membrane was exposed overnightat -80° C. in a cassette in which an X-ray film (Kodak) was placed toprepare an autoradiograph.

From the pattern of the obtained autoradiograph, 10 phages wereclassified into 3 types. Of DNA fragments inserted into these 3 types ofphage vectors, the DNA fragment, which was detected when the azuki beanEXT gene was used as the probe, underwent subcloning to the plasmidvector to analyze a partial nucleotide sequence. The result indicatedthat the DNA fragment inserted into phage vectors of all plaquescontained a gene analogous to the EXT gene, namely a family gene.However, the EXT gene was not contained therein.

EXAMPLE 6

Cloning of DNA Fragment Containing "The Promoter Downstream" Region ofAzuki Bean EXT Gene from Azuki Bean Partial Genome DNA Library

The genome DNA extracted from azuki bean leaves in the same manner as inExample 5 was subjected to digestion with restriction enzymes of EcoR Iand Hind III, double digestion with EcoR I-Hind III, and 0.7% agarosegel electrophoresis, followed by transfer to a nylon membrane andSouthern hybridization using, as a probe, the azuki bean EXT gene cDNAprepared in the same manner as in Example 5-(3).

The Southern hybridization was carried out according to the methoddescribed in Example 5-(3), except that, after the hybridization, themembrane was washed thrice with a washing solution containing 2×SSC and0.1% SDS at room temperature for 20 minutes, washed twice with the samewashing solution at 50° C. for 20 minutes, and then washed twice with awashing solution containing 0.1×SSC and 0.1% SDS at 50° C. for 20minutes.

The result revealed that about 3 bands were detected on each lane andthe most intense band appeared for the EcoR I digest at about 8.5 kbp,for the Hind III at about 8.5 kbp, and for the EcoR I-Hind IIIdouble-digest at about 5.5 kbp. Then, 30 μg of DNA that was completelydouble-digested with EcoR I-Hind III was subjected to 0.7% agarose gelelectrophoresis, recovery of a band around about 5.5 kbp from theagarose gel, ligation to the EcoR I-Hind III site of λEXlox (Novagene),and packaging by using in-vitro Packaging Kit (Stratagene), followed byinfection with E. coli ER1647, the host bacterium, to obtain a partialazuki bean genome DNA library of a size centered with the about 5.5 kbpDNA fragment having the EcoR I-Hind III site at both termini. The titerof this library was 1.9×10⁶ pfu/ml.

Next, plaque hybridization using the azuki bean EXT gene cDNA as a probewas carried out in the same manner as in Example 5. Ten positive plaqueswere obtained from 1.3×10⁵ plaques. These plaques were suspended in a SMbuffer solution and each plaque underwent secondary screening. In thesecond screening, the above-mentioned azuki bean EXT gene cDNA as wellas an oligonucleotide VAN-U7 (Sequence ID NO 17), synthesized on thebasis of a 5'-noncoded region having a low homology with an isozyme ofthe azuki bean EXT gene cDNA, were utilized as a probe, respectively. Inthe case where the azuki bean EXT gene cDNA was utilized as the probe,the plaque hybridization and washing were carried out in the same manneras described above. In the case where the synthetic oligonucleotideVAN-U7 was utilized, the hybridization probe was prepared by labelingwith [γ-³² P]ATP using the 5'-Terminal-Labeling Kit MEGALABEL™ (TAKARASHUZO Co., Ltd.) at the 5'-terminus. The specific activity of this probewas about 2×10⁸ cpm/μg. The plaque hybridization using this probe wascarried out in the same manner as in Example 2, except that thepre-hybridization and the hybridization were carried out at 47° C. Afterthe hybridization, the membrane was washed twice with a washing solutioncontaining 6×SSC and 0.1% SDS at room temperature and then washed twicewith the same washing solution at 47° C. for 20 minutes. The resultrevealed that, of 10 positive plaques, 4 plaques were DNA fragmentscontaining the EXT gene cDNA. The phages inserted with these fragmentswere subjected to automatic subcloning by infection with E. coli BM25.8,a host bacterium having the PlCre gene, where a region subclonedautomatically in the host was converted to a pUC-type plasmid. Thethus-prepared plasmid was named as pVXG303.

The E. coli JM109 strain (TAKARA SHUZO Co., Ltd.) transformed withpVXG303 is denoted & imprinted as Escherichia coli JM109/pVX303 and hasbeen deposited on Mar. 15, 1995 (the date of original deposit) inNational Institute of Bioscience and Human-Technology, Agency ofIndustrial Science and Technology, Ministry of Industrial Trade andIndustry (1-3, Higashi 1-Chome, Tsukuba-Shi, Ibaragi-ken, 305, Japan) asthe accession No. FERN BP-5390, in accordance with the Budapest Treaty.This plasmid underwent nucleotide sequencing of the DNA fragmentsaccording to the Sanger method using BcaBEST™ Dideoxy Sequencing Kit(TAKARA SHUZO Co., Ltd.). Partial nucleotide sequences thereof are shownin SEQ ID NO 18 and SEQ ID NO 19 in the Sequence Listing. Comparison ofthese sequences with the sequence of the azuki bean EXT gene cDNArevealed that said fragment contained a promoter of the azuki bean EXTgene.

EXAMPLE 7

Cloning of DNA Fragment Containing Promoter Region of Azuki Bean EXTGene by Inverse PCR

(1) Examination of Self-Ligation Efficiency

pVXG303 in Example 6 is a plasmid of about 9.5 kbp having an EcoR I/HindIII fragment originating from a genome DNA of 5.5 kbp containing apromoter region of the azuki bean EXT gene.

As a control for self-ligation and inverse PCR, this plasmid wasdigested with restriction enzyme Hind III and then self-ligated at DNAconcentrations of 10 ng/μl, 3.3 ng/μl, 2 ng/μl, and 1 ng/μl,respectively. After the ligation, 5 μl each of the samples wastransformed into E. coli JM109 and the self-ligation efficiency wasobtained from the number of colonies formed. The result indicated thatthe self-ligation efficiency increased with diluting the DNAconcentration. However, in the case where polymerase chain reactions(PCRs) with these ligation solutions used as templates were carried outby using a primer VAN-UH1 (SEQ ID NO 21) as a sense primer and a primerVAN-L (SEQ ID NO 22) as an antisense primer, an inhibition was inducedwhen a larger volume of the ligation solution was added in the reactionsystem in order to increase the template amount and also the recoverydecreased with diluting the DNA concentration when ethanol precipitationwas carried out in order to decrease the template amount in the reactionsystem. These results revealed that the objective cyclic DNA could beobtained efficiently when the DNA concentration and the reaction volumein the ligation were adjusted at 3.3 ng/μl and 300 μl, respectively.

(2) Inverse PCR with Hind III Fragment of Azuki Bean DNA Used asTemplate

One μg of the genome DNA prepared from azuki bean leaves in the samemanner as in Example 5 was completely digested with restriction enzymeHind III, extracted once with the phenol/chloroform solution todeactivate the enzyme, and then underwent ethanol precipitation. Theethanol-precipitated DNA was mixed with 268 μl of distilled water, 30 μlof a 10× ligation buffer solution, and 2 μl of T4 DNA Ligase (TAKARASHUZO Co., Ltd.) and then underwent self-ligation by reaction at 16° C.overnight. With 0.1 μg of the obtained cyclic genome DNA used as thetemplate, PCR using TaKaRa LA PCR Kit (TAKARA SHUZO Co., Ltd.) wascarried out by using primer VAN-UH1 (SEQ ID NO 21) as a sense primer andprimer VAN-L (SEQ ID NO 22) as an antisense primer. The reaction wascarried out by repeating a cycle of 94° C. (0.5 minute), 55° C. (1minute), and 72° C. (2 minutes) 30 times. However, any amplification wasnot observed in this reaction. Then, with 1 μl of this reaction solutionused as a template, PCRs were carried in the same manner with repeatingthe above-mentioned cycle 30 times by using:

1) primer VAN-UH2 (SEQ ID NO 23) as a sense primer and primer VAN-L6(SEQ ID NO 24) as an antisense primer,

2) primer VAN-UH3 (SEQ ID NO 25) as a sense primer and primer VAN-L3(SEQ ID NO 26) as an antisense primer,

3) primer VAN-UH2 (SEQ ID NO 23) as a sense primer and primer VAN-L3(SEQ ID NO 26) as an antisense primer, and

4) primer VAN-UH3 (SEQ ID NO 25) as a sense primer and primer VAN-L6(SEQ ID NO 24) as an antisense primer.

Analyses of the reaction solutions after the reactions by 1% agarose gelelectrophoresis revealed that a DNA fragment of about 1.8 kbp wasamplified specifically in the combination of 3). It was difficult inother combinations to identify the objective fragments owing to theamplification of many nonspecific DNA fragments.

The DNA fragment obtained in the primer combination of 3) was recoveredfrom the gel and subjected to end-blunting using DNA Blunting Kit(TAKARA SHUZO Co., Ltd.), phosphorylation of the PCR product using the5'-Terminal-Labeling Kit MEGALABEL™ (TAKARA SHUZO Co., Ltd.) at the5'-terminus, and then subcloning at the Hinc II site of pUC119 (TAKARASHUZO Co., Ltd.). Three plasmids were selected therefrom and underwentnucleotide sequencing of the DNA fragments according to the Sangermethod using BcaBEST™ Dideoxy Sequencing Kit (TAKARA SHUZO Co., Ltd.).Since partial nucleotide sequencing indicated that the nucleotidesequences were identical for all plasmids, the total nucleotide sequencewas determined by using one of these sequences.

A partial nucleotide sequence thereof is shown in SEQ ID NO 27 in theSequence Listing. Also, the restriction map of said DNA fragment isshown in FIG. 2. The plasmid containing said DNA fragment is denoted aspVXP-H3, whereas E. coli JM109 strain transformed with said pVXP-H3 isdenoted and indicated as Escherichia coli JM 109/pVXP-H3 and has beendeposited on Feb. 17, 1995 (the date of original deposit) in NationalInstitute of Bioscience and Human-Technology, Agency of IndustrialScience and Technology, Ministry of Industrial Trade and Industry (1-3,Higashi 1-Chome, Tsukuba-Shi, Ibaragi-ken, 305, Japan) as the accessionNo. FERM BP-5388, in accordance with the Budapest Treaty.

EXAMPLE 8

Cloning of DNA Fragment Containing Promoter Upstream Region of AzukiBean EXT Gene from Azuki Bean Genome DNA Library

(1) Construction of Genomic DNA Library

Conditions were examined in order to subject the genome DNA obtained inExample 5 to partial digestion with restriction enzyme Sau3A I. That isto say, 10 U/μl of Sau3A I (TAKARA SHUZO Co., Ltd.) was diluted with adiluent buffer solution to adjust its concentration in a 50 μl reactionsolution (1 μg DNA) to 1 to 0.035 U/μg DNA, which was reacted at 37° C.for 30 minutes and then mixed with 1 μl of 0.5 M EDTA to stop thereaction. After the reaction, a 20 μl sample was analyzed by 0.4%agarose gel electrophoresis to indicate that molecules of 15 to 20 kbpsize were formed most abundantly under the condition with 0.1 U/μg DNA.The reaction was scaled up under this condition.

Next, 160 μg of DNA, partially digested under this condition, wasutilized for attempted fractionation of molecules of 15-20 kbp in sizes,by carrying out NaCl-density gradient centrifugation. That is to say,density gradients of 1.25 to 5 M NaCl were prepared into centrifugetubes fitted to a HITACHI RPS40-T Rotor, 200 μl each of DNA (about 160μg) was placed slowly, and ultracentrifugation using a HITACHI SCP70Hultracentrifuge was carried out at 35000 rpm for 3 hours. After thecentrifugation, samples were divided in Eppendorf tubes with 250 μleach. Analysis of 20 μl aliquots taken from every 3 tubes by 0.4%agarose gel electrophoresis indicated that fractions Nos. 18 to 21seemingly contained DNA molecules of appropriate sizes. Therefore, eachof 0.3 μg, 0.6 μg, and 1.2 μg of the DNA from fraction No. 20 was mixedwith a 1.0 μg solution of λGEM11 Arm (Promega Biotech) to make an 8 μlsolution, which, after addition of 8 μl of Solution II and 16 μl ofSolution I in TaKaRa Ligation Kit Version 2 (TAKARA SHUZO Co., Ltd.),underwent ligation at 26° C. for 10 minutes. Each of the samples afterthe ligation was subjected to twice extraction with phenol and ethanolprecipitation. Then, the total amount underwent packaging by using an invitro packaging kit (Stratagene), followed by infection with E. coliLE392, the host bacterium, to obtain an azuki bean genome DNA library.The titer of this library was 1.1×10⁵ pfu/ml.

(2) Screening of Library

The genome DNA fragment obtained in Example 6 was labeled with [α-³²P]dCTP using BcaBEST™ Labeling Kit (TAKARA SHUZO Co., Ltd.) to prepare aprobe for hybridization. The plaque hybridization using this probe wascarried out on the above-prepared genome DNA library in the same manneras in Example 5. After the hybridization, the membrane was washed thricewith a washing solution containing 2×SSC and 0.1% SDS at roomtemperature for 20 minutes and further washed once with a washingsolution containing 1×SSC and 0.1% SDS at 50° C. for 20 minutes. Phageswere inoculated on 20 square plates so as to form plaques at 1×10⁴plaques per plate. As a result of screening on 2×10⁵ phages obtainedfrom a total of 20 plates, 21 positive plaques were obtained. Next, eachof these plaques underwent secondary screening in order to isolate itspositive clone respectively.

The secondary screening was carried out by inoculation of a phagesolution, which was taken from each of 21 positive plaques obtained inthe primary screening, as thinly as possible so as to form about 300plaques per a square plate, followed by plaque hybridization. In thesecond screening, the genomic DNA fragment obtained in Example 6 in thesame manner as in the primary screening as well as an oligonucleotideVAN-U7 (SEQ ID NO 17), synthesized on the basis of a 5'-noncoded regionhaving a low homology with family genes such as other isozymes (theazuki bean EXT2 and the azuki bean EXT3) in the azuki bean EXT gene cDNAsequence, respectively were utilized as a probe. In the case where thegenomic DNA fragment was utilized as the probe, the plaque hybridizationand washing were carried out in the same manner as in the firstscreening. In the case where the synthetic oligonucleotide VAN-U7 wasutilized, the same procedure as in the case of Example 6 was applied.

Of 10 plaques showing positive signals obtained in the secondaryscreening, one plaque with an intense signal was selected for carryingout tertiary screening. However, this plaque with an intense signal wasonly one in about 1000 plaques even in the secondary screening.Furthermore, this only plaque was very small. For the purpose ofpurified proliferation of this plaque, the tertiary screening wascarried out by inoculation on 6 circular plates (90 mm φ) so as to form100 to 200 plaques per a plate. They were utilized for the hybridizationand washing in the same manner as in the secondary screening. As aresult, an extremely small plaque of a needle-tip size was detected at aposition that did not correspond to the plaque recognized as the signalat first glance but corresponded to the signal on a very carefulobservation of the plate. A phage DNA was extracted using this plaque bya careful application of the plate lysate method. DNA fragments insertedinto a phage vector of said plaque were extracted and then a DNAfragment of about 11 kbp in length was obtained.

By carrying out double digestion of this DNA fragment with EcoR I-HindIII and Southern hybridization using, as a probe, the azuki bean EXTgene genome DNA fragment obtained in Example 6, a shorter DNA fragmentcontaining a promoter region of this gene could be defined. Nucleotidesequencing using the plasmid inserted with this DNA fragment andcomparison of said fragment with the sequence of the azuki bean EXT genecDNA revealed whether this DNA fragment contained a promoter of theazuki bean EXT gene. FIG. 1 shows the restriction map of said fragment.Also, a partial nucleotide sequence of said fragment is shown in SEQ IDNO 20 in the Sequence Listing. The plasmid integrated with this fragmentinto the EcoR I and Hind III sites of pUC118 (TAKARA SHUZO Co., Ltd.) isdenoted as pVXP11, whereas E. coli JM109 strain transformed with pVXP101is denoted and indicated as Escherichia coli JM109/pVXP101 and has beendeposited on Feb. 23, 1995 (the date of original deposit) in NationalInstitute of Bioscience and Human-Technology, Agency of IndustrialScience and Technology, Ministry of Industrial Trade and Industry (1-3,Higashi 1-Chome, Tsukuba-Shi, Ibaragi-ken, 305, Japan) as the accessionNo. FERM BP-5389, in accordance with the Budapest Treaty.

EXAMPLE 9

Northern Hybridization Using Azuki Bean Young Plant

(1) Preparation of Total RNA

Tissues taken from stems, buds, cotyledons, and leaves of 5 day-old and40 day-old azuki bean plants after seeding were frozen in liquidnitrogen collectively and then kept at -80° C. until RNA extraction wasoperated. Total RNAs were extracted from each of these frozen tissues bythe guanidine thiocyanate/phenol method. That is to say, 1 g of frozencells was placed in a tube containing 2.5 ml of a guanidine thiocyanatesolution [a 200-ml solution prepared by dissolving 100 g of guanidinethiocyanate and 1.47 g of sodium citrate dihydrate in water is kept at4° C. and 7 μl of mercaptoethanol and 5 mg of sodium lauroylsarcosinateper 1 ml are added before use], crushed with a Polytron to effectextraction, mixed with 2.5 ml of a phenol-chloroform-isoamyl alcohol(25:24:1) mixture, stirred gently for 15 minutes, and then centrifugedat 3000 rpm for 10 minutes. Then, the separated aqueous layer was mixedwith 2.5 ml of a phenol-chloroform-isoamyl alcohol (25:24:1) mixturewith vigorous stirring and the resulting suspension was centrifuged toseparate an aqueous layer. This procedure was repeated twice. Next, theresulting aqueous layer was mixed with 2.0 ml of aphenol-chloroform-isoamyl alcohol (25:24:1) mixture with vigorousstirring and the resulting suspension was centrifuged to separate anaqueous layer, which was mixed with 3 M sodium acetate and ethanol, andthen centrifuged to obtain an RNA precipitate. This precipitate wascompletely dissolved in 2 ml of a Tris-SDS solution [50 mM Tris-HCl (pH:9.0) and 1% SDS] and placed in a tube containing 2 ml of water-saturatedphenol, which was shaken well. The resulting suspension was centrifugedto separate an aqueous layer, to which 2 ml of water-saturated phenoland 2 ml of a chloroform-isoamyl alcohol (49:1) mixture weresuccessively added with vigorous stirring and the resulting suspensionwas centrifuged to separate an aqueous layer. Next, the resultingaqueous layer was mixed with 2 ml of a chloroform-isoamyl alcohol (49:1)mixture with vigorous stirring and the resulting suspension wascentrifuged to separate an aqueous layer, which was mixed with 3 Msodium acetate and ethanol, and then centrifuged to obtain an RNAprecipitate. This precipitate was completely dissolved in 0.5 ml ofsterilized water and the concentration was adjusted to 1 mg/ml bymeasuring the absorbance. The resulting solution was mixed with a 1/4volume of 10 M lithium chloride with stirring, allowed to stand at 4° C.for 2 hours, and then centrifuged to obtain a precipitate. Thisprecipitate was completely dissolved in 1 ml of sterilized water, mixedwith 3 M sodium acetate and ethanol, and then centrifuged to obtainabout 0.6 mg of an RNA precipitate.

(2) Northern Hybridization

A fragment of the azuki bean EXT gene cDNA [EP-0562836 A1 (1993)] waslabeled with [α-³² P]dCTP using BcaBEST™ Labeling Kit (TAKARA SHUZO Co.,Ltd.) to prepare a probe for northern hybridization.

The northern hybridization was carried out in the following wayaccording to the method described in "Molecular Cloning, A laboratoryManual", Second Edition, Chapter 7, pp. 7.39-7.52 (T. Maniatis et al.,published by Cold Spring Harbor Laboratory Press in 1989). That is tosay, the extracted total RNA was subjected to electrophoresis withformaldehyde-running agarose gel (1%), followed by neutralization in anammonium acetate solution and northern blotting on a nylon membrane(Hybond-N) overnight. After RNA was fixed by irradiation with aultraviolet transilluminator (254 nm) for 5 minutes, the membrane wassubjected to pre-hybridization in 20 ml of a pre-hybridization buffersolution (50% formaldehyde, 0.65 M NaCl, 0.1 M Na-PIPES (pH: 6.8),5×Denhardt's solution, 0.1% SDS, 5 mM EDTA, and 100 μg/ml salmon-spermDNA] at 42° C. for 3 hours. Then, the ³² P-labeled probe prepared by theabove-mentioned method was added to 20 ml of a pre-hybridization buffersolution [50% formaldehyde, 0.65 M NaCl, 0.1 M Na-PIPES (pH: 6.8),5×Denhardt's solution, 0.1% SDS, 5 mM EDTA, and 10% dextran sulfate]. Tothis probe solution was added the membrane obtained by thepre-hybridization and hybridization was carried out at 42° C. overnight.

After the hybridization, the membrane was washed thrice with a washingsolution containing 2×SSC and 0.1% SDS at 50° C. for 20 minutes. Afterbeing dried, the membrane was exposed overnight at -80° C. in a cassettein which an X-ray film (Kodak) was placed to prepare an autoradiograph.

The result revealed that the EXT gene expression was observedspecifically in stems and the gene was expressed particularly in a partthat was grown with elongation.

EXAMPLE 10

Northern Hybridization Using Tobacco Cultured Cells

(1) Preparation of Total RNA

Tobacco BY2 cultured cells, which were cultivated for 1, 4, 6, 8, and 10days, respectively were collected on a Buchner filter funnel by suctionfiltration. At this time, the suction was applied for additional 10 to30 seconds after the culture medium was filtered out on the funnel, soas to remove the liquid culture medium completely. After the culturemedium was drained off, about 1 g of cells was quickly recovered byweighing, immediately frozen in liquid nitrogen, and then kept at -80°C. until RNA extraction was operated. The frozen cells were placed in atube containing 2 ml of an extraction solution [200 mM Tris-HCl (pH:9.0), 100 mM NaCl, 10 mM EDTA, 0.5% SDS, and 14 mM 2-mercaptoethanol]and 2 ml of water-saturated phenol, crushed with a Polytron for 5minutes to effect the extraction, mixed with 2 ml of achloroform-isoamyl alcohol (49:1) mixture, and vigorously stirredfurther with a Polytron. The resulting suspension was centrifuged toseparate an aqueous layer. Next, the resulting aqueous layer wassuccessively mixed with 2 ml of water-saturated phenol and 2 ml of achloroform-isoamyl alcohol (49:1) mixture with vigorous stirring and theresulting suspension was centrifuged to separate an aqueous layer. Thisprocedure was repeated twice. The resulting aqueous layer was mixed with2 ml of a chloroform-isoamyl alcohol (49:1) mixture with vigorousstirring and the resulting suspension was centrifuged to separate anaqueous layer, which was mixed with 3 M sodium acetate and ethanol, andthen centrifuged to obtain about 0.7 mg of an RNA precipitate.

(2) Northern Hybridization

The tobacco EXT gene cDNA (JP 7-79778 A) and a cDNA fragment (SEQ ID NO16) of the family gene tobacco XRP1 described in Example 4, respectivelywere labeled with [α-³² P]dCTP using BcaBEST™ Labeling Kit (TAKARA SHUZOCo., Ltd.) to prepare a probe for northern hybridization.

The northern hybridization was carried out in the same way as the methoddescribed in Example 9-(2). The results are shown in FIG. 3. That is tosay, FIG. 3 illustrates the expressions of EXT and XRP, wherein theexpression of a tobacco EXT mRNA was shown in the upper row, theexpression of a tobacco XRP mRNA was shown in the middle row, and therRNA amounts were shown in the lower row.

As can be seen from FIG. 3, it was revealed that the expression of thetobacco EXT gene was observed on the first day of the cultivation,reaching to a peak on the 4th day. Conversely, the tobacco XRP1 gene, afamily gene of the EXT gene shown in Example 4, was expressed intenselyon the first day of the cultivation and after the 6th day.

At the same time, the growth curve for the tobacco BY2 culture cells wasalso drawn by measuring the number of cells and the packed cell volume(PCV). The cell number was obtained by treatment of the tobacco BY2culture cells with an enzyme solution (pH: 5.5) containing 1%cellulase-ONOZUKA (Yakult Honsha Co., Ltd.), 0.1% pectolyase Y23(SEISHIN Corporation), and 0.4 M mannitol at 30° C. for 2 hours to beconverted into cell wall-free protoplasts, followed by counting thenumber of the protoplasts with a blood counter. Furthermore, PCV wasobtained by centrifugation of a culture suspension (10 ml) of thetobacco BY2 culture cells, taken in a 15 ml, graduated centrifuge tube,at 2000 rpm for 5 minutes by using a swing rotor, followed bymeasurement of the volume of cell pellets. Hereupon, a mean value (n=5)was plotted on the graph shown in FIG. 4. That is to say, FIG. 4illustrates the growth in the tobacco BY2 cell culture, wherein thevertical axes represent PCV (%) and the cell number, and the horizontalaxis represents the time (day).

The results illustrated in FIGS. 3 and 4 indicated that the tobacco EXTgene was expressed in any time and the expression was intenseparticularly in an early period of the logarithmic growth phase.

It was also indicated that the tobacco XRP1 gene, a family gene of theEXT gene shown in Example 4, was expressed intensely in the inductionphase and the stationary phase.

EXAMPLE 11

Transient Assay Using Tobacco Culture Cells

(1) Construction of Plasmid for Transfer

Using pBI121 (Clontech) having the cauliflower mosaic virus 35Spromoter, the E. coli-origin GUS gene, and a transcription terminationsequence cassette originating from nopaline synthetase, the EcoR site ofthis plasmid was first removed by subjecting said plasmid toend-blunting by using DNA Blunting Kit (TAKARA SHUZO Co., Ltd.) aftercomplete digestion with restriction enzyme EcoR I and transformationinto E. coli JM 109 strain after self-ligation. The obtained plasmid wasnamed as pB1221EL and E. coli JM 109 strain transformed with pB1221ELwas named as Escherichia. coli JM 109/pB1221EL. In order to remove thecauliflower mosaic virus 35S promoter region in the plasmid, thisplasmid was subjected to digestion with restriction enzymes Hind III andXba I and then purification of the objective fragment other than the 35Spromoter region by agarose gel electrophoresis followed by cutting-off.

Next, with pVXG303 prepared in Example 6 used as the template, PCR wascarried out by using primer VAN-UHE (SEQ ID NO 29) as a sense primer andprimer VAN-LX (SEQ ID NO 30) as an antisense primer. The reaction wascarried out by repeating a cycle of 94° C. (1 minute), 55° C. (2minutes), and 72° C. (3 minutes) 10 times. The resulting fragment wassubjected to recovery by separation with 2.5% agarose gelelectrophoresis, ligation into the Hind III and Xba I sites of theabove-mentioned pB1221EL, and transformation into E. coli JM 109 strain.This plasmid was named as pEXTΔEGUS and E. coli JM 109 straintransformed with pEXTΔEGUS was named as Escherichia. coli JM109/pEXTΔEGUS.

Next, pEXTΔEGUS was subjected to end-blunting by using DNA Blunting Kit(TAKARA SHUZO Co., Ltd.) after complete digestion with restrictionenzyme Hind III. The resulting DNA fragment was subjected to completedigestion with restriction enzyme EcoR I, terminal dephosphorylation byBAP treatment, and purification by agarose gel electrophoresis.

On the other hand, pVXP-H3 prepared in Example 7 was subjected toend-blunting after complete digestion with restriction enzyme Xba I(TAKARA SHUZO Co., Ltd.), followed by complete digestion withrestriction enzyme EcoR I.

An about 960-bp DNA fragment containing a promoter region of the Xba Isite to the EcoR I site of the azuki bean EXT gene was subjected topurification by agarose gel electrophoresis, ligation with theabove-mentioned pEXTΔEGUS DNA fragment, and transformation into E. coliJM 109 strain. This plasmid was named as pEXTΔXGUS and E. coli JM 109strain transformed with pEXTΔXGUS was named as Escherichia. coli JM109/pEXTΔXGUS.

PEXTΔEGUS was subjected to complete digestion with restriction enzymesHind III and EcoR I, ligation with a DNA fragment containing a promoterobtained by complete digestion of pVXP-H3, prepared in Example 7, withrestriction enzymes Hind III and EcoR I, and then transformation into E.coli JM 109 strain. This plasmid was named as pEXTGUS and E. coli JM 109strain transformed with pEXTGUS was named as Escherichia. coli JM109/pEXTGUS.

(2) Gene Transfer by Electroporation

The electroporation method was applied to the transfer into tobacco BY2culture cells by each of pEXTΔEGUS, pEXTΔXGUS, and pEXTGUS, prepared asdescribed above, as well as by each of promoter-free pBI101 (Clontech;denoted as pGUS in FIG. 5) having only the GUS gene cassette and pBI221(Clontech) having the cauliflower mosaic virus 35S promoter and the GUSgene, used as controls.

First, the tobacco BY2 culture cells were treated with an enzymesolution (pH: 5.5) containing 1% cellulase-ONOZUKA (Yakult Honsha Co.,Ltd.), 0.1% pectolyase Y23 (SEISHIN Corporation), and 0.4 M mannitol at30° C. for 2 hours to be converted into cell-wall-free protoplasts. Asuspension of the 2×10⁶ protoplasts of the tobacco BY2 culture cells inan electroporation buffer solution (70 mM KCl, 5 mM MES, and 0.3 Mmannitol, pH 5.8) was mixed with 3 pmol of each plasmid DNA and a 10%.PEG 6000/electroporation buffer solution with stirring. An electricpulse (300 V, 125 μF) using Gene Pulser II (Bio-Rad Laboratories) wasapplied to the resulting mixture to transfer the DNA into the plantcells.

The cells were incubated in the Linsmaier-Skoog culture medium[Physiologia Plantarum, 18, 100 (1965)] containing 0.2 mg/l 2,4-D as anauxin, 1% sucrose, and 0.4 M mannitol at 26° C. for 40 hours after thetransfer. The cells were recovered by extraction and a mixture of therecovered cells in 200 μl of an extraction buffer solution [50 mMphosphate buffer (pH 7.0), 10 mM EDTA, 0.1% Triton X-100, 0.1% Sarkosyl,and 10 mM 2-mercaptoethanol] placed in an Eppendorf tube was subjectedto ultra-sonication on ice for 30 seconds by using a ultrasonicatorW-225 (Heatsystems-Ultrasonics) with setting the output control at 1.5and the duty cycle at 50%. Then, a supernatant isolated bycentrifugation was used for the assay of the GUS activity and the assayof the protein quantity.

(3) Measurement of Promoter Activity

The reaction was carried out by adding 45 μl of the extraction buffersolution and 25 μl of a 4 mM 4-MUG substrate to each 30 μl of theextract placed in a 96-well microtiter plate for fluorescence. After 5,35, and 95 minutes, the reaction was terminated by addition of 50 μl ofa reaction-termination solution (1 M Na₂ CO₃). Then, the specificfluorescence emitted by 4-MU, the reaction, product, at an excitationwavelength of 365 nm and fluorescence wavelength of 455 nm, was measuredwith a fluorescence plate reader [Fluoroscan II (Labosystems)].

Moreover, the protein quantity was assayed by a procedure described asfollows. Thus, 2, 5, 10, 15, 20, and 30 μl of a 1/5-diluted solution ofthe extract or an 800 μg/ml BSA standard solution (20 μl of the extractbuffer solution is mixed with 80 μl of 1 mg/ml BSA) were placed in a96-well microtiter plate and thereto were added respectively 158, 155,150, 145, 140, and 130 μl of distilled water and 40 μl of the assayreagent in Bio-Rad Protein Assay Kit (Bio-Rad Laboratories). After beingstirred slowly and then allowed to stand for 20 minutes at roomtemperature, the mixture was measured by a plate reader (wavelength: 590nm) within 60 minutes to assay the amount of protein.

The GUS activity was measured in the following way. At the same timewhen the above assays were carried out, the fluorescence intensities ofthe 4-MU standard solutions were measured and the results were plottedon a graph with the 4-MU quantity (pmol) at the x-axis and thefluorescence intensity at the y-axis. Then, the 4-MU quantity per onefluorescence unit was obtained from the slope and, further, the resultson the samples were plotted on a graph with the time (minute) at thex-axis and the fluorescence intensity at the y-axis to obtain theincreasing rate of the fluorescence intensity and then to obtain thedecomposition rate of 4-MUG equal to the GUS activity. In addition, theGUS specific activity was obtained from the amount of protein. Theresults are shown in FIG. 5. In other words, FIG. 5 illustrates themeasurement of the EXT promoter activity using the transformed tobaccoBY2 culture cells, wherein the bar graph in the figure shows theGUS-specific activity (pmol 4MU/minute/mg protein) upon the transfer ofeach plasmid and the restriction map of the promoter region of eachplasmid is illustrated thereunder.

As shown in FIG. 5, it could be verified that the DNA fragmentcontaining the EXT gene promoter region exhibited an activity moreintense than that of the cauliflower mosaic virus 35S promoter that hadbeen said to be expressed intensely in the plants.

EXAMPLE 12

Detection of Tissue Specificity Using Transformed Arabidopsis

(1) Construction of Plasmids for Transfer

In order to obtain plasmids for the transfer, as shown in FIGS. 6 and 7,a binary vector pBI-HI-35SIG [Plant and Cell Physiology, 31, 805-813(1990)] having a transcription termination sequence cassette originatingfrom nopaline synthetase and, as a marker gene, a gene resistant tohygromycin (HPT) and kanamycin (NPTII), and a GUS gene containing an E.coli-origin intron and the cauliflower mosaic virus 35S promoter,respectively, were digested with restriction enzymes Hind III and SnaB I(TAKARA SHUZO Co., Ltd.), and then purified by cutting out the objectivefragment other than the 35S promoter region by agarose electrophoresis.Then, each of pEXTGUS prepared in Example 11 and above-mentionedPEXTΔXGUS were digested with restriction enzymes Hind III and SnaB I,and then purified by cutting out the fragment containing the azuki beanEXT promoter region by agarose gel electrophoresis. These fragmentsrespectively were subjected to ligation at the Hind III and SnaB I sitesof the above-mentioned pBI-HI-35SIG, and then transformation into E.coli JM 109 strain. These plasmids for the transfer were named aspBVEG101 and pBVEG121, respectively, and E. coli JM 109 strainstransformed with these plasmids were named as Escherichia. coli JM109/pBVEG101 and Escherichia. coli JM 109/pBVEG121, respectively.

Furthermore, as shown in FIGS. 8 and 9, the Hind III and SnaB Ifragments of promoter-free pBI101 (Clontech) having only the GUS genecassette and pBI121 (Clontech) having the cauliflower mosaic virus 35Spromoter were subcloned be the same procedure as described above at theHind III and SnaB I sides of pBI-HI-35SIG to obtain the plasmids forcontrol experiments. The thus-obtained plasmids were named as pBI-H-101and pBI-H-121, respectively, and E. coli JM 109 strains transformed withthese plasmids were named as Escherichia. coli JM 109/pBI-H-101 andEscherichia. coli JM 109/pBI-H-121, respectively.

(2) Transformation of Agrobacterium for Infection

Each of the above-mentioned plasmids was mixed with Agrobacteriumtumefaciens EHA101 competent cells [SHOKUBUTU SAIBOU KOUGAKU (Plant CellTechnology) 4 (3), 193-203 (1992)], emitted with an electric pulse (2.5kV, 25 μF, 200 Ω) using Gene Pulser II (Bio-Rad Laboratories), andcultivated at 30° C. for 2 days to transfer the plasmid into theAgrobacterium strain. The Agrobacterium strains transformed with theseplasmids were named as Agrobacterium tumefaciens EHA101/pBVEG101,Agrobacterium tumefaciens, EHA101/pBVEG121, Agrobacterium tumefaciensEHA101/pBI-H-101, and Agrobacterium tumefaciens EHA101/pBI-H-121,respectively.

(3) Production of Transgenic Plants

WS seeds, an eco-type of Arabidopsis thaliana, (available fromNotlingham Arabidopsis Stock Center: NASC) were disinfected on thesurface with 20% hypochlorite, then sowed on an MSO plate(MURASHIGE-Skoog inorganic salt mixture (WAKO Pure Chemicals Industries,Ltd.), mixed with 2% sucrose, 3 mg/l thiamine hydrochloride, 5 mg/lnicotinic acid, and 0.5 mg/l pyridoxine hydrochloride, is adjusted to pH6.3, mixed further with 0.2% gellan gum, autoclaved, and plated],underwent low-temperature treatment at 4° C. for 2 days, and thencultivated at 22° C. under continuous irradiation of a 3000-lux light.Transplantation on a new MSO plate was carried out at 1 week and 2 weeksafter the sowing, respectively, and on 2 days after the transplantationat 2 weeks, 3 to 4 stumps were bundled and cut to prepare about 1cm-long sections of the roots. The sections of the roots were placedside by side on a CIM plate (0.5 mg/l 2,4-dichlorophenoxyacetic acid and0.05 mg/l kinetin are added to the MSO plate) and cultivated at 22° C.for 2 days under continuous irradiation of a 3000 lux light. Thereafter,the sections of the 2 day-cultivated roots were soaked for 30 seconds ina solution prepared by cultivation of each of the Agrobacterium strainsobtained in (2) at 30° C. for 2 days followed by 5-fold dilution withthe MS solution, soaked up to remove excess water, placed side by sideon a new CIM plate, and then cultivated for 2 days. Two days later, theinfected sections were transferred on a SIMC plate [to the MSO plate areadded 5 mg/l N6-(2-isopentenyl)adenine, 0.15 mg/l indoleacetic acid, and0.3 g/l carbenicillin], cultivated for 2 days, and then transplanted ona SIMCS plate (to the SIMC plate are added 50 mg/l of kanamycin and 20mg/l of hygromycin). The plants were repeatedly transplanted on a newSIMCS plate once or twice per every week.

When regeneration of shoots were observed and the regenerated plantswere equipped with complete rosette leaves of about 5 mm, the plantparts were cut off from the callus, and lightly inserted on a RIM plate(0.5 mg/l indoleactic acid is added to the MSO plate). Each of rootedplants underwent final transplanting on rock wool and cultivation in aliquid [Hyponecks (Hyponecks Japan) is diluted 1000-fold with water] toobtain T2 seeds.

(4) Detection of Tissue Specificity

The seeds obtained in (3) were sown on an MSKH plate (50 mg/l kanamycinand 20 mg/l hygromycin are added to the MSO plate) to select resistantstocks. The resistant stocks underwent final transplanting on rock wooland cultivation in a liquid [Hyponecks (Hyponecks Japan) is diluted1000-fold with water].

A sample was collected by cutting off a portion of the ground part ofplants that flowered and initiated silique formation, after about 30days from the sowing. The cut plant sections were soaked in a fixedsolution (20% paraformaldehyde, 0.1 M phosphate buffer, 1 mM EDTA, pH7.0) at room temperature for 1 hour, washed twice with 0.1 M phosphatebuffer, and then soaked in a substrate solution [2 mM X-Gluc, 50 mMphosphate buffer (pH 7.0), 0.5% Triton X-100, and 20% methanol]. Afterdeaeration for 25 minutes to facilitate penetration of the substratesolution, the reaction was carried out at 37° C. for 1-3 days. After thereaction, the sample was washed with 70% ethanol, then observed bysoaking into 40% glycerol, and reserved.

The results of the reaction indicated that a GUS-specific stain was notobserved in wild-type plants as well as in those transferred withpBI-H-101 whereas the stain was detected in all tissues of plantstransferred with pBI-H-121 (the cauliflower mosaic virus 35S promoter).

On the other hand, in plants transferred with pBVEG101 and pBVEG121containing the azuki bean EXT gene promoter, the GUS stain was observedat an elongation part of stem, at the tips of leave and silique, and atthe tip of pistil, indicating that these portions possessed a potentpromoter activity. Of these results, the results on wild-type,pBI-H-121, and pBVEG101 were illustrated in FIG. 10.

EXAMPLE 13

Isolation of Azuki Bean EXT2 Gene Promoter by Inverse PCRs with HindIII, Nsp V, and Xba I Fragments of Azuki Bean Genome DNA Used asTemplates

One μg of the genome DNA prepared from azuki bean leaves in the samemanner as in Example 5 placed in separate tubes was completely digestedwith each of restriction enzymes Hind III, Nsp V, and Xba I,respectively, extracted once with the phenol/chloroform solution todeactivate the enzyme, and then underwent ethanol precipitation. Theethanol-precipitated DNA was mixed with 268 μl of distilled water, 30 μlof a 10×ligation buffer solution, and 2 μl of T4 DNA Ligase (TAKARASHUZO Co., Ltd.) and then underwent self-ligation by reaction at 16° C.overnight. With 0.1 μg of the obtained cyclic genome DNA used as thetemplate, PCR using TAKARA LA PCR Kit (TAKARA SHUZO Co., Ltd.) wascarried out by using primer IP44-3 (SEQ ID NO 31) as a sense primer andprimer IP44-5 (SEQ ID NO 32) as an antisense primer. The reaction wascarried out by repeating a cycle of 94° C. (1 minute), 98° C. (20seconds), and 67° C. (10 minutes) 30 times, finally followed by 72° C.(10 minutes). After the reaction, 5 μl of the reaction solutionunderwent 1% agarose gel electrophoresis, indicating that an about 6.0kbp band was observed only in the sample digested with restrictionenzyme Hind III. For other samples, any amplification was not observedin this reaction. Then, 1 μl each of a 100-fold dilution of the sampledigested with restriction enzyme Hind III and other reaction solutionwithout dilution was used as a template for PCR that was carried out inthe same manner by using primer IP44-2 (SEQ ID NO 33) as a sense primerand primer IP44-5 (SEQ ID NO 32) as an antisense primer. After thereaction, 5 μl of the reaction solution underwent analysis by 1% agarosegel electrophoresis, confirming that the about 6.0 kbp band was veryintense and thus was amplified specifically in the sample digested withrestriction enzyme Hind III. In the sample digested with restrictionenzyme Nsp V, a large number of seemingly nonspecific DNA fragments wereamplified, with an about 1. 2-kbp band being likely a main band. In thesample digested with restriction enzyme Xba I, a large number ofseemingly nonspecific DNA fragments were amplified and thusidentification of the objective fragment was difficult. Then, from thesample digested with restriction enzyme Hind III, a DNA fragmentobtained in the primary PCR was recovered from the gel and subjected toend-blunting using DNA Blunting Kit (TAKARA SHUZO Co., Ltd.),phosphorylation of the PCR product using the 5'-Terminal-Labeling KitMEGALABEL™ (TAKARA SHUZO Co., Ltd.) at the 5'-terminus, and thentransfer into the Hinc II site of pUC118. The resulting plasmid wastransformed into E. coli JM109, but no colonies were obtained.

Then, it was planned that the restriction map of the about 6.0-kbp PCRfragment was prepared to define the promoter region and then severalfragments were separated and subcloned.

First, an about 3.1 kbp band and an about 2.9 kbp band were separated byend-blunting of this PCR fragment followed by digestion with restrictionenzyme Hind III. These DNA fragments were subjected together to ligationto the Hind III-Hinc II site of pUC118 and transformation into E. coliJM109, but only a plasmid containing a fragment inserted with the about2.9 kbp DNA was obtained and that with the about 3.1 kbp DNA was notobtained. In addition, the results on PCR using primer IP44-2 (SEQ ID NO33) and M13 Primer M4 (TAKARA SHUZO Co., Ltd.) as well as on PCR usingprimer IP44-6 (SEQ ID NO 34) and M13 Primer M4 (TAKARA SHUZO Co., Ltd.)revealed that amplification occurred only for primer IP44-6 (SEQ ID NO34) and M13 Primer M4 and that the 2.9 kbp inserted fragment contained a3'-downstream region of the azuki bean EXT2 gene whereas the 3.1 kbpfragment contained the promoter region.

FIG. 11 illustrates the restriction map of the about 6.0 kbp PCRfragment amplified with primers IP44-3 and IP44-5. In the figure, theupper part in the restriction map corresponds to the nucleotide sequenceof primer IP44-5 and the lower part corresponds to the nucleotidesequence of primer IP44-3.

Next, because of the existence of two EcoR I sites on this about 6.0 kbpfragment, the PCR fragment was subjected to end-blunting followed bydigestion with restriction enzymes Hind III and EcoR I to separate anabout 0.4 kbp band, an about 0.5 kbp band, and an about 2.55 kbp band.Since the restriction map (FIG. 11) indicates the existence of promoterregions at about 0.5, kbp and at 2.55 kbp, each of these bands wassubjected to ligation to the EcoR I-Hinc II site and the EcoR I-Hind IIIsite of pUC118, followed by transformation into E. coli JM109.

Of colonies thus obtained, 16 colonies from the ligation at the EcoRI-Hinc II site were screened by PCR using primer IP44-2 (SEQ ID NO 33)and M13 Primer M4, (TAKARA SHUZO Co., Ltd.), revealing that 7 colonieswere positive. Of these positive colonies, plasmids were extracted from3 colonies and were named as pVX2P501, pVX2P503, and pVX2P505,respectively.

The nucleotide sequences of inserted fragments contained in pVX2P501,pVX2P503, and pVX2P505 were determined by subjecting each of pVX2P501,pVX2P503, and pVX2P505 to the sequence analysis of respective insertedfragment portions according to the Sanger method using M3 Primer M4(TAKARA SHUZO Co., Ltd.) and M13 Primer RV (TAKARA SHUZO Co., Ltd.),followed by comprehensive interpretation of there results. Comparison ofthese sequences with the sequence (SEQ ID NO 11) of the original azukibean EXT2 cDNA revealed that the overlapping portions were identical andalso 3 types of clones had the completely identical sequence.

Forty six colonies from the ligation at the EcoR I-Hind III site werescreened by PCR using primer IP44-2 (SEQ ID NO 33) and M13 Primer M4(TAKARA SHUZO Co., Ltd.), revealing that 27 colonies contained an about2.6 kbp inserted fragment.

Next, when this about 2.6 kbp fragment was digested with restrictionenzyme Acc I (TAKARA SHUZO Co., Ltd.), an about 600 bp fragment appearedfrom 3 colonies and an about 500 bp fragment appeared from 12 colonies.Since the afore-mentioned restriction map (FIG. 11) indicates that anabout 600 bp fragment appears from clones containing promoter regions,plasmids were extracted from 3 positive colonies and were named aspEXT2pro(F)f1, pEXT2pro(F)f2, and pEXT2pro(F)f3, respectively.

The partial sequence analysis of each of pEXT2pro(F)f1, pEXT2pro(F)f2,and pEXT2pro(F)f3 according to the Sanger method using M13 Primer M4(TAKARA SHUZO Co., Ltd.) and M13 Primer RV (TAKARA SHUZO Co., Ltd.)revealed that 3 types of clones had the completely identical sequence.Next, in order to sequence the entire region of the about 2.6 kbpinserted fragment, a Pst I site adapter, which was prepared by using asynthetic oligomer E/Psite (1) (SEQ ID NO 35) and a synthetic oligomerE/Psite (2) (SEQ ID NO 36) was transferred into the EcoR I site ofpEXT2pro(F)f3. This transfer allowed to transfer only the Pst I site atthe side opposite to the EcoR I site of pEXT2pro(F)f3.

In addition, after complete digestion of this plasmid with restrictionenzymes Pst I and EcoR I, Kilo-Sequence Deletion Kit (TAKARA SHUZO Co.,Ltd.) was utilized to obtain clones that were deleted between the EcoR Isite and the inserted fragment side.

The nucleotide sequences of inserted fragments contained inpEXT2pro(F)f3 were determined by subjecting some selected, deletedclones of appropriate lengths to the sequence analysis of respectiveinserted fragment portions according to the Sanger method using M13Primer M4 (TAKARA SHUZO Co., Ltd.) and M13 Primer RV (TAKARA SHUZO Co.,Ltd.), followed by comprehensive interpretation of there results.

The fact that the nucleotide sequences of inserted fragments containedin pVX2P501, pVX2P503, and pVX2P505 are continuous on the genome withthe nucleotide sequences of inserted fragments contained inpEXT2pro(F)f3 was confirmed by PCRs and direct sequencing of boundaryregions thereof. SEQ ID NO 3 in the Sequence Listing shows an about 3.0kbp sequence in the promoter region upstream from this azuki bean EXT2N-terminal amino acid sequence.

EXAMPLE 14

Isolation of Azuki Bean EXT3 Gene Promoter by Inverse PCRs with HindIII, Nsp V, and Xba I Fragments of Azuki Bean Genome DNA Used asTemplates

With 0.1 μg of the cyclic genome DNA, prepared by complete digestion ofan azuki bean genome DNA using restriction enzymes Hind III, Nsp V, andXba I, followed by self-ligation, in the same manner as described inExample 13, used as the template, PCR using TaKaRa LA PCR Kit (TAKARASHUZO Co., Ltd.) was carried out by using primer IP45-3 (SEQ ID NO 37)as a sense primer and primer IP45-5 (SEQ ID NO 38) as an antisenseprimer. The reaction was carried out by repeating a cycle of 94° C. (1minute), 98° C. (20 seconds), and 67° C. (10 minutes) 30 times, finallyfollowed by 72° C. (10 minutes). After the reaction, 5 μl of thereaction solution underwent 1% agarose gel electrophoresis, indicatingthat an about 4.5 kbp band was observed only in the sample digested withrestriction enzyme Nsp V. For other samples, any amplification was notobserved in this reaction. Then, 1 μl each of a 100-fold dilution of thesample digested with restriction enzyme Nsp V and other reactionsolution without dilution was used as a template for PCR that wascarried out in the same manner by using primer IP45-2 (SEQ ID No 39) asa sense primer and primer IP45-6 (SEQ ID NO 40) as an antisense primer.After the reaction, 5 μl of the reaction solution underwent analysis by1% agarose gel electrophoresis, confirming that the about 4.5 kbp bandwas very intense and thus was amplified specifically in the sampledigested with restriction enzyme Nsp V. In the sample digested withrestriction enzyme Hind III, a large number of seemingly nonspecific DNAfragments were amplified and thus identification of the objectivefragment was difficult. Furthermore, in the sample digested withrestriction enzyme Xba I, two main bands of about 4.5 kbp and about 3.5kbp were identified.

Then, from the sample digested with restriction enzyme Nsp V, an about4.5-kbp DNA fragment obtained in the primary PCR was recovered from thegel and subjected to end-blunting using DNA Blunting Kit (TAKARA SHUZOCO., Ltd.), phosphorylation of the PCR product using the5'-Terminal-Labeling Kit MEGALABEL™ (TAKARA SHUZO Co., Ltd.) at the5'-terminus, and then transfer into the Hinc II site of pUC118. Theresulting plasmid was transformed into E. coli JM109.

Of colonies obtained, 46 colonies were screened by PCR using primerIP45-2 (SEQ ID NO 39) and M13 Primer 14 (TAKARA SHUZO Co., Ltd.),revealing that 3 colonies were positive. Of these positive colonies,plasmids were extracted from 3 colonies and were named as pVX3P206,pVX3P234, and pVX3P237, respectively.

Each of pVX3P206, pVX3P234, and pVX3P237 was subjected to the sequenceanalysis of the both terminal portions of respective, inserted fragmentsaccording to the Sanger method using M13 Primer M4 (TAKARA SHUZO Co.,Ltd.) and M13 Primer RV (TAKARA SHUZO Co., Ltd.). The results indicatedthat these sequences contained the primers used in the PCR andcomparison of these sequences with the sequence (SEQ ID NO 12) of theoriginal azuki bean EXT3 cDNA revealed that the overlapping portionswere identical. Also, 3 types of clones had the completely identicalsequence in the range analyzed.

When this fragment was completely digested with restriction enzyme NspV, the about 4.0 kbp band was separated into two bands of about 0.5 kbpand about 3.5 kbp. Then, PCR method was used to identify which of thetwo bands of about 0.5 kbp and about 3.5 kbp contained the promoterregion. The results revealed that the about 0.5 kbp band was the DNAfragment containing the promoter region.

Then, it was planned that the two main bands of about 4.5 kbp and about3.5 kbp, obtained by the secondary PCR of the sample digested withrestriction enzyme Xba I, were used to clone the 5'-upstream of saidpromoter region.

When these DNA fragments were completely digested with restrictionenzyme Nsp V, the about 4.5 kbp band was separated into two bands ofabout 0.5 kbp and about 4.0 kbp. On the other hand, the about 3.5 kbpband was not digested with restriction enzyme Nsp V. Accordingly, theabout 4.5 kbp band was considered to contain the promoter region.

Then, the restriction map of the about 4.5 kbp DNA fragment was preparedby digestion of the about 4.5 kbp DNA fragment with restriction enzymesNsp V and Xba I, followed by double digestion with restriction enzymesNsp V-Xba I.

FIG. 12 illustrates the restriction map of the about 4.5 kbp fragmentamplified with primers IP45-3 and IP45-5. In the figure, the upper partin the restriction map corresponds to the nucleotide sequence of primerIP45-5 and the lower part corresponds to the nucleotide sequence ofprimer IP45-3.

As a result, it was revealed that the about 3.0 kbp DNA fragment of NspV-Xba I was the 5'-upstream of the promoter region.

Then, the 3.0 kbp DNA fragment formed by double digestion of the about4.5 kbp DNA fragment with restriction enzymes Nsp V-Xba I was recoveredfrom the gel and transferred into the Xba I-Acc I site of pbluescript SK(-) (Stratagene). The resulting plasmid was transformed into E. coliJM109.

Of the obtained colonies, 7 colonies were screened by PCR using M13Primer M4 (TAKARA SHUZO Co., Ltd.) and M13 Primer RV (TAKARA SHUZO Co.,Ltd.), revealing that 6 colonies contained the about 3.0 kbp insertedfragment. Plasmids were extracted from these 6 colonies and named aspVX3P101, pVX3P103, pVX3P104, pVX3P105, pVX3P106, and pVX3P107,respectively.

Of these plasmids, each of pVX3P101, pVX3P103, pVX3P104, and pVX3P107were subjected to the partial sequence analysis of the nucleotidesequence of respective, inserted fragment portion according to theSanger method using M13 Primer M4 (TAKARA SHUZO Co., Ltd.) and M13Primer RV (TAKARA SHUZO Co., Ltd.), revealing that 4 types of clones hadthe completely identical sequence. Next, in order to sequence the entireregion of the about 3.0 kbp inserted fragment, after complete digestionof pVX3P107 with restriction enzymes Kpn I (TAKARA SHUZO Co., Ltd.) andXho I (TAKARA SHUZO Co., Ltd.), Kilo-Sequence Deletion Kit (TAKARA SHUZOCo., Ltd.) was utilized to obtain clones that were deleted between theXho I site and the inserted fragment side. The nucleotide sequences ofinserted fragments contained in pVX3P107 were determined by subjectingsome selected, deleted clones of appropriate lengths to the sequenceanalysis of respective, inserted fragment portions according to theSanger method using M13 Primer M4 (TAKARA SHUZO Co., Ltd.) and M13Primer RV (TAKARA SHUZO Co., Ltd.), followed by comprehensiveinterpretation of there results. The fact that the nucleotide sequencesof inserted fragments contained in pVX3P206, pVX3P234, and pVX3P237 arecontinuous on the genome with the nucleotide sequences of insertedfragments contained in pVX3P107 was confirmed by PCRs and directsequencing of boundary regions thereof. SEQ ID NO 4 in the SequenceListing shows an about 3.4 kbp sequence in the promoter region upstreamfrom the thus-obtained azuki bean EXT3 N-terminal amino acid sequence.

EXAMPLE 15

Isolation of Azuki Bean XRP1 Gene Promoter by Inverse PCRs with HindIII, Nsp V, and Xba I Fragments of Azuki Bean Genome DNA Used asTemplates

With 0.1 μg of the cyclic genome DNA, prepared by complete digestion ofan azuki bean genome DNA using restriction enzymes Hind III, Nsp V, andXba I, followed by self-ligation, in the same manner as described inExample 13, used as the template, PCR using TAKARA LA PCR Kit (TAKARASHUZO Co., Ltd.) was carried out by using primer IPM6-3 (SEQ ID NO 41)as a sense primer and primer IPM6-4 (SEQ ID NO 42) as an antisenseprimer.

The reaction was carried out by repeating a cycle of 94° C. (1 minute),98° C. (20 seconds), and 67° C. (10 minutes) 30 times, finally followedby 72° C. (10 minutes). After the reaction, 5 μl of the reactionsolution underwent 1% agarose gel electrophoresis, indicating that anyamplification was not observed.

Then, 1 μl of the reaction solution was used as a template for PCR thatwas carried out in the same manner by using primer IPM6-2 (SEQ ID NO 43)as a sense primer and, primer IPM6-5 (SEQ ID NO 44) as an antisenseprimer. After the reaction, 5 μl of the reaction solution underwentanalysis by 1% agarose gel electrophoresis, confirming that in thesample digested with restriction enzyme Hind III and Nsp V, a largenumber of seemingly nonspecific DNA fragments were amplified and thusidentification of the objective fragment was difficult. Furthermore, inthe sample digested with restriction enzyme Xba I, two main bands ofabout 2.5 kbp and about 0.6 kbp were identified.

Then, from the sample digested with restriction enzyme Xba I, two DNAfragments of about 2.5 kbp and about 0.6 kbp obtained in the secondaryPCR were recovered from the gel and subjected to end-blunting using DNABlunting Kit (TAKARA SHUZO Co., Ltd.), phosphorylation of the PCRproduct using the 5'-Terminal-Labeling Kit MEGALABEL™ (TAKARA SHUZO Co.,Ltd.) at the 5'-terminus, and then transfer into the Hinc II site ofpUC118. The resulting plasmid was transformed into E. coli JM109.

Of each group of colonies obtained, 6 colonies were respectivelyscreened by PCR using M13 Primer M4 (TAKARA SHUZO Co., Ltd.) and M13Primer RV (TAKARA SHUZO Co., Ltd.), revealing that 5 colonies werepositive from the about 2.5 kbp band and 3 colonies were positive fromthe about 0.6 kbp band. Plasmids were extracted from these positivecolonies. The plasmids from the about 2.5 kbp band were named aspXRG301, pXRG302, pXRG303, pXRG304, and pXRG305, respectively. Also, theplasmids from the about 0.6 kbp band were named as pXRG403, pXRG404, andpXRG406, respectively.

Complete digestion of pXRG301, pXRG302, pXRG303, pXRG304, and pXRG305with both restriction enzymes EcoR I and Sph I (TAKARA SHUZO Co., Ltd.)revealed that only three plasmids from pXRG302, pXRG303, and pXRG304 hadan about, 2.5 kbp inserted fragment. Furthermore, complete digestion ofpXRG301, pXRG302, pXRG303, pXRG304, pXRG305, pXRG403, pXRG404, andpXRG406 with restriction enzyme Xba I resulted in cleavage at one sitein the inserted fragment other than one site in the vector to form anabout 1.1 kbp band, whereas pXRG403, pXRG404, and pXRG406 were cleavedonly at the site existing in the vector. These results suggested thatpXRG301, pXRG302, pXRG303, pXRG304, and pXRG305 contained the objectiveazuki bean XRP1 promoter region.

Each of pXRG301, pXRG302, pXRG303, and pXRG304 was subjected to thesequence analysis of the both terminal portions of respective, insertedfragments according to the Sanger method using M13 Primer M4 (TAKARASHUZO Co., Ltd.) and M13 Primer RV (TAKARA SHUZO Co., Ltd.). The resultsindicated that the sequences of pXRG302 and pXRG303 contained theprimers used in the PCR and comparison of these sequences with thesequence (SEQ ID NO 14) of the original azuki bean XRP1 cDNA revealedthat the overlapping portions were identical. Also, 2 types of cloneshad the completely identical sequence.

FIG. 13 illustrates the restriction map of the about 2.5 kbp fragmentamplified with primers IPM6-2 and IPM6-5. In the figure, the upper partin the restriction map corresponds to the nucleotide sequence of primerIPM6-5 and the lower part corresponds to the nucleotide sequence ofprimer IPM6-2.

The nucleotide sequence of an azuki bean XRP1 promoter region of about1.1 kbp in inserted fragments contained in pXRG302 was determined by thesequence analysis of respective, inserted fragment portions according tothe Sanger method using M13 Primer M4 (TAKARA SHUZO Co., Ltd.) and M13Primer RV (TAKARA SHUZO Co., Ltd.), followed by further analysis of aprimer synthesized on the basis of the sequence in the insertedfragments and comprehensive interpretation of there results. SEQ ID NO 5in the Sequence Listing shows an about 1.1 kbp sequence in the promoterregion upstream from this azuki bean XRP1 N-terminal amino acidsequence.

EXAMPLE 16

Isolation of Tomato Gene Promoter by Inverse PCRs with EcoR I, Hind III,Nsp V, and Xba I Fragments of Tomato Genome DNA Used as Templates

Seeds of Lycopersicon esculentum cv. Ponterosa (TAXII SEED Co., Ltd.)were germinated and then cultivated for about one month to obtain about10 g of leaves and stems. About 2.5 g of these leaves and stems werepulverized in a mortar in the presence of liquid nitrogen to prepare awhite powder. The resulting leave and stem powder was immediately placedin a 50 ml polystyrene tube and extracted with 10 ml of a urea-phenolDNA extraction buffer solution [0.05 M Tris-HCl (pH: 7.6), 0.02 M EDTA,5% phenol, 8 M urea, 0.35 M NaCl, and 2% sodium lauroylsarcosinate]mixed with 25% SDS at 65° C. for 1 hour. The extract was mixed with a2-fold volume of a phenolchloroform-isoamyl alcohol (25:24:1) mixture,stirred gently for about 15 minutes, and then centrifuged at 2000 rpmfor 15 minutes. After the centrifugation, the supernatant wastransferred into a new tube, again mixed with a 2-fold volume of aphenol-chloroform-isoamyl alcohol (25:24:1) mixture, stirred gently forabout 15 minutes, and then centrifuged at 2000 rpm for 15 minutes. Thesupernatant after this centrifugation was transferred into a new tube,mixed with a 2-fold volume of ethanol, and stirred gently. Then, theprecipitated, white genome DNA was coiled out by using a Pasteur pipetand transferred into a new tube. To this tube was added 1.5 ml of a TEbuffer solution [10 mM Tris-HCl (pH: 8.0) and 1 mM EDTA] and theresulting mixture was kept at 55° C. overnight to dissolve the DNA.Analysis of 1 μl of a sample, prepared by diluting of this DNA solution10-fold, by 0.4% agarose gel electrophoresis revealed that the solutioncontained a high molecular DNA at a concentration of about 100 ng/μl. Inother words, about 150 μg of the genomic DNA was obtained from about 2.5g of the plant portions.

One μg of this genomic DNA was taken in separate tubes and subjected tocomplete digestion using restriction enzymes EcoR I, Hind III, Nsp V,and Xba I, respectively, followed by self-ligation, in the same manneras described in Example 13. With 0.1 μg of the thus-prepared cyclicgenomic DNA used as the template, PCR using TAKARA LA PCR Kit (TAKARASHUZO Co., Ltd.) was carried out by using primer IPLE-3 (SEQ ID NO 45)as a sense primer and primer IPLE-4 (SEQ ID NO 46) as an antisenseprimer. The reaction was carried out by repeating a cycle of 94° C. (1minute), 98° C. (20 seconds), and 67° C. (10 minutes) 30 times, finallyfollowed by 72° C. (10 minutes). After the reaction, 5 μl of thereaction solution underwent 1% agarose gel electrophoresis, indicatingthat an about 6.6-kbp band was observed in the sample digested withrestriction enzymes Hind III and Xba I. For other samples, anyamplification was not observed in this reaction. Then, with 1 μl of thereaction solution, obtained from the sample digested with restrictionenzyme Xba I, used as the template, secondary PCR using TAKARA LA PCRKit (TAKARA SHUZO Co., Ltd.) was carried out by using primer IPLE-2 (SEQID NO 47) as a sense primer and primer IPLE-5 (SEQ ID NO 48) as anantisense primer. The reaction was carried out under the same conditionsas described above by repeating the cycle 10 times. After the reaction,the obtained DNA fragments were recovered from the gel and thentransferred into pT7Blue T-Vector (Novagen). The resulting plasmids weretransformed into E. coli JM109.

Of the obtained colonies, 12 colonies were screened by PCR using TAKARALA PCR Kit (TAKARA SHUZO Co., Ltd.) was carried out by using primerIPLE-1 (SEQ ID NO 49) and primer IPLE-6 (SEQ ID NO 50), indicating that6 colonies were positive. Plasmids were extracted from these 6 coloniesand named as pLXG101, pLXG102, pLXG103, pLXG106, pLXG109, and pLXG110,respectively.

Each of pLXG101, pLXG102, and pLXG106 was subjected to the sequenceanalysis of respective, inserted fragments according to the Sangermethod using M13 Primer M4 (TAKARA SHUZO Co., Ltd.) and M13 Primer RV(TAKARA SHUZO Co., Ltd.).

Comparison of these sequences with the sequence [EP-0562836 A1 (1993)]of the original tomato EXT cDNA revealed that the overlapping portionswere identical. However, 2 to 3 base substitution was detected withinthe sequenced range. It was conceived that this substitution involved amistake induced by the nested PCR in the polymerase reaction.Accordingly, after the promoter region in one of the 6 plasmids wassequenced, a primer was synthesized and other clones were alsosequenced, thereby inferring the sequence on the actual genome.

FIG. 14 illustrates the restriction map of the about 6.6 kbp insertedfragment amplified with primers IPLE-2 and IPLE-5. In the figure, theupper part in the restriction map corresponds to the nucleotide sequenceof primer IPLE-5 and the lower part corresponds to the nucleotidesequence of primer IPLE-2.

Moreover, complete digestion of pLXG101, pLXG102, pLXG103, pLXG106,pLXG109, and pLXG110 with restriction enzyme Xba I, followed by agaroseelectrophoresis, revealed formation of two bands around about 4.9 kpbfor pLXG101, pLXG102, pLXG103, and pLXG110 as well as two bands at about8.1 kbp and at about 1.7 kbp for pLXG106 and pLXG109. This observationindicated that PCR-amplified DNA fragments were inserted in the reversedirections for three plasmids of pLXG101, pLXG102, pLXG103, and pLXG110and for two plasmids of pLXG106 and pLXG109. In addition, it wasrevealed from the results on PCR using TAKARA LA PCR Kit (TAKARA SHUZOCo., Ltd.), which was carried out, with these plasmids used astemplates, by using M13 Primer M4 (TAKARA SHUZO Co., Ltd.) and primerIPLE-1 (SEQ ID NO 49), that the about 4.9 kbp DNA fragment contained thetomato EXT gene promoter region, among the DNA fragments of about 4.9kbp and about 1.7 kbp which were separated upon complete digestion ofthe inserted fragment with restriction enzyme Xba I.

Then, pLXG106 was subjected to complete digestion with restrictionenzyme Xba I, followed by ethanol precipitation. Theethanol-precipitated DNA was mixed with 268 μl of distilled water, 30 μlof a 10×ligation buffer solution, and 3 μl of T4DNA Ligase (TAKARA SHUZOCo., Ltd.) and then underwent self-ligation by reaction at 16° C.overnight. The resulting plasmids were transformed into E. coli JM109.

Of the obtained colonies, plasmids were extracted from 4 colonies andnamed as pLXG601, pLXG602, pLXG603, and pLXG604, respectively. Doubledigestion of these pLXG601, pLXG602, pLXG603, and pLXG604 withrestriction enzymes EcoR I-Pst I, followed by agarose electrophoresis,revealed that the about 4.9 kbp inserted fragment existed in all ofthese plasmids.

Furthermore, since the above-mentioned restriction map (FIG. 14) hasindicated the existence of one Hind III site in the about 4.9 kbpinserted fragment, pLXG106 was subjected to complete digestion withrestriction enzyme Hind III, followed by addition of 268 μl of distilledwater, 30 μl of a 10×ligation buffer solution, and 3 μl of T4DNA Ligase(TAKARA SHUZO Co., Ltd.) to the ethanol-precipitated DNA and thenself-ligation by reaction at 16° C. overnight. The resulting plasmidswere transformed into E. coli JM109.

Of the obtained colonies, plasmids were extracted from 6 colonies andnamed as pLXP101, pLXP102, pLXP103, pLXP106, pLXP109, and pLXP111,respectively. Double digestion of these pLXP101, pLXP102, pLXP103,pLXP106, pLXP109, and pLXP111 with restriction enzymes EcoR I-Pst I,followed by agarose electrophoresis, revealed that the about1.4-kbp-inserted fragment existed in all of these plasmids.

pLXP101 was subjected to the sequence analysis of the nucleotidesequence of the inserted fragment portion according to the Sanger methodusing M13 Primer M4 (TAKARA SHUZO Co., Ltd.) and M13 Primer RV (TAKARASHUZO Co., Ltd.).

Next, in order to sequence the entire region of the about1.4-kbp-inserted fragment in pLXP101, after complete digestion ofpLXP101 with restriction enzymes Kpn I and BamH I, Kilo-SequenceDeletion Kit (TAKARA SHUZO Co., Ltd.) was utilized to obtain clones thatwere deleted between the BamH I site and the inserted fragment side. Thenucleotide sequences of inserted fragments contained in pLXP101 weredetermined by subjecting some selected, deleted clones of appropriatelengths to the sequence analysis of respective, inserted fragmentportions according to the Sanger method using M13 Primer M4 (TAKARASHUZO Co., Ltd.) and M13 Primer RV (TAKARA SHUZO Co., Ltd.), followed bycomprehensive interpretation of there results. In addition, the about1.4 kbp promoter region in pLXG102 and pLXG103, using the primerssynthesized on the basis of the nucleotide sequence of thepLXP101-inserted fragment, was sequenced and compared, thereby inferringthe sequence for the about 1.4 kbp promoter region in the upstream fromthe tomato EXT gene N-terminal amino acid sequence. This sequence isshown in SEQ ID NO 6 in the Sequence Listing.

EXAMPLE 17

Isolation of Tobacco EXT Gene Promoter by Inverse PCRs with EcoR I, HindIII, Nsp V, and Xba I Fragments of Tobacco Genome DNA Used as Templates

About 150 mg of the tobacco BY2 culture cells (callus) was pulverized ina mortar to a powder, which was mixed with 0.5 ml of an extractionsolution [15% sucrose, 50 mM Tris-HCl (pH 8.0), and 50 mM EDTA],transferred into an Eppendorf tube, and centrifuged at 500 rpm for 1minute. The precipitate was dissolved in 300 μl of 2T-1E [20 mM Tris-HCl(pH 8.0) and 10 mM EDTA], mixed with 40 μl of 10% SDS, shaken slowly,and then treated at 70° C. for 15 minutes. The resulting solution wasmixed with 225 μl of 5 M ammonium acetate, stirred, placed on ice for 30minutes, and centrifuged at 15000 rpm for 15 minutes. Aftercentrifugation, the supernatant was transferred into a new tube, mixedwith 0.7 m21 of isopropanol, stirred, allowed to stand at roomtemperature for 15 minutes, and centrifuged at 15000 rpm for 15 minutes.After the supernatant was removed, the residue was mixed with ice-cold80% ethanol and centrifuged at 15000 rpm for 15 minutes. The precipitatewas dried and mixed with 100 μl of a TE buffer solution [10 mM Tris-HCl(pH: 8.0) and 1 mM EDTA], and the resulting mixture was kept at 4° C.overnight to dissolve the DNA. Analysis of 5 μl of the DNA solution by0.4% agarose gel electrophoresis revealed that the solution contained ahigh molecular DNA at a concentration of about 100 ng/μl. In otherwords, about 10 μg of the genomic DNA was obtained from about 150 mg ofthe callus.

One μg of this genomic DNA was taken in separate tubes and subjected tocomplete digestion using restriction enzymes EcoR I, Hind III, Nsp V,and Xba I, respectively, followed by self-ligation, in the same manneras described in Example 13. With 0.1 μg of the thus-prepared cyclicgenome DNA used as the template, PCR using TAKARA LA PCR Kit (TAKARASHUZO Co., Ltd.) was carried out by using primer IPTE-3 (SEQ ID NO 51)as a sense primer and primer IPTE-4 (SEQ ID NO 52) as an antisenseprimer. The reaction was carried out by repeating a cycle of 94° C. (1minute), 98° C. (20 seconds), and 67° C. (10 minutes) 30 times, finallyfollowed by 72° C. (10 minutes). After the reaction, 5 μl of thereaction solution underwent 1% agarose gel electrophoresis, indicatingthat an about 1.2 kbp band was observed in the sample digested withrestriction enzyme Xba I. For other samples, any amplification was notobserved in this reaction. Then, with 1 μl of the above reactionsolution, obtained from the sample digested with restriction enzyme XbaI, used as the template, secondary PCR using TAKARA LA PCR Kit (TAKARASHUZO Co., Ltd.) was carried out by using primer IPTE-2 (SEQ ID NO 53)as a sense primer and primer IPTE-5 (SEQ ID NO 54) as an antisenseprimer. The reaction was carried out under the same conditions asdescribed above. As a result, a DNA fragment of about 1.1 kbp wasamplified. The DNA fragments obtained by the secondary PCR wererecovered from the gel and then transferred into pT7Blue T-Vector(Novagen). The resulting plasmids were transformed into Nova BlueCompetent Cells (Novagen).

Of the obtained colonies, 12 colonies were screened by PCR using primerIPTE-1 (SEQ ID NO 55) and primer IPTE-6 (SEQ ID NO 56), indicating thatall 12 colonies were positive. Of them, plasmids were extracted from 6colonies and named as pNXG101, pNXG102, pNXG103, pNXG104, pNXG105, andpNXG106, respectively.

Each of pNXG102, pNXG103, and pNXG104 was subjected to the sequenceanalysis of respective, inserted fragments according to the Sangermethod using M13 Primer M4 (TAKARA SHUZO Co., Ltd.) and M13 Primer RV(TAKARA SHUZO Co., Ltd.). Comparison of these sequences with thesequence (JP 7-79778 A) of the original tobacco EXT cDNA revealed thatthe overlapping portions were completely identical.

FIG. 15 illustrates the restriction map of the about 1.1 kbp insertedfragment amplified with primers IPTE-2 and IPTE-5. In the figure, theupper part in the restriction map corresponds to the nucleotide sequenceof primer IPTE-5 and the lower part corresponds to the nucleotidesequence of primer IPTE-2.

Moreover, complete digestion of pNXG101, pNXG102, pNXG103, pNXG104,pNXG105, and pNXG106 with restriction enzyme Xba I, followed by agaroseelectrophoresis, revealed formation of two bands at about 3.1 kbp and atabout 0.9 kbp for pNXG101 as well as two bands at about 3.8 kbp and atabout 0.2 kbp for pNXG102, pNXG103, pNXG104, pNXG105, and pNXG106. Thisobservation indicated that the EXT was inserted in the reversedirections for pNXG101 and for pNXG102, pNXG103, pNXG104, pNXG105, andpNXG106. In addition, it was revealed from the results on PCR, which wascarried out, with these plasmids used as templates, by using M13 PrimerM4 (TAKARA SHUZO Co., Ltd.) and primer IPTE-1 (SEQ ID NO 55) or primerIPTE-6 (SEQ ID NO 56), that the about 0.9 kbp DNA fragment contained thetobacco EXT gene promoter region, among the DNA fragments of about 0.9kbp and about 0.2 kbp which were separated upon complete digestion ofthe inserted fragment with restriction enzyme Xba I.

Then, pNXG103 was subjected to complete digestion with restrictionenzyme Hind III, followed by ethanol precipitation. Theethanol-precipitated DNA was mixed with 268 μl of distilled water, 30 μlof a 10×ligation buffer solution, and 3 μl of T4 DNA Ligase (TAKARASHUZO Co., Ltd.) and then underwent self-ligation by reaction at 16° C.overnight. The resulting plasmids were transformed into E. coli JM109.Plasmids were extracted from 3 colonies and named as pT-EXT-4, pT-EXT-5,and pT-EXT-6, respectively. Double digestion of these pT-EXT-4,pT-EXT-5, and pT-EXT-6 with restriction enzymes Hind III-EcoR I,followed by agarose electrophoresis, revealed that the about 0.4 kbpinserted fragment existed in all of these plasmids. These pT-EXT-4,pT-EXT-5, and pT-EXT-6 were subjected to the sequence analysis of thenucleotide sequence of the inserted fragment portion according to theSanger method using M13 Primer M4 (TAKARA SHUZO Co., Ltd.) and T7Promoter Primer (Novagen).

Furthermore, pNXG102 was completely digested with restriction enzymesHind III and Xba I, and an about 0.5 kbp band was cut out by agarose gelelectrophoresis for purification.

Next, this DNA fragment was ligated to the molecule obtained by doubledigestion of pUC18 (TAKARA SHUZO Co., Ltd.) with restriction enzymesHind III-Xba I. The resulting plasmids were transformed into E. coliJM109.

Of the obtained colonies, plasmids were extracted from 3 colonies andnamed as pT-EXT-1, pT-EXT-2, and pT-EXT-3, respectively. Doubledigestion of these pT-EXT-1, pT-EXT-2, and pT-EXT-3 with restrictionenzymes Hind III-EcoR I, followed by agarose electrophoresis, revealedthat the about 0.5 kbp inserted fragment existed in all of theseplasmids.

These pT-EXT-1, pT-EXT-2, and pT-EXT-3 were subjected to the sequenceanalysis of the nucleotide sequence of the inserted fragment portionaccording to the Sanger method using M13 Primer M4 (TAKARA SHUZO Co.,Ltd.) and M13 Primer RV (TAKARA SHUZO Co., Ltd.).

On the basis of comprehensive interpretation of there results, theentire nucleotide sequence in the promoter region upstream from thistobacco EXT N-terminal amino acid sequence was determined. This sequenceis shown by SEQ ID NO 7 in the Sequence Listing.

EXAMPLE 18

Isolation of Wheat Gene Promoter by Inverse PCRs with EcoR I, Hind III,Nsp V, and Xba I Fragments of Wheat Genome DNA Used as Templates

One μg of a wheat genome DNA (Clontech) was taken in separate tubes andsubjected to complete digestion using restriction enzymes EcoR I, HindIII, Nsp V, and Xba I, respectively, followed by self-ligation, in thesame manner as described in Example 13. With 0.1 μg of the thus-preparedcyclic genome DNA used as the template, PCR using TAKARA LA PCR Kit(TAKARA SHUZO Co., Ltd.) was carried out by using primer KOM-1 (SEQ IDNO 57) as a sense primer and primer KOM-4 (SEQ ID NO 58) as an antisenseprimer in the reaction system with a total volume of 50 μl. The reactionwas carried out by repeating a cycle of 94° C. (1 minute), 98° C. (20seconds), and 67° C. (10 minutes) 30 times, finally followed by 72° C.(10 minutes). After the reaction, 5 μl of the reaction solutionunderwent 1% agarose gel electrophoresis, indicating that an about 4.3kbp band and an about 3.5 kbp band were observed in the sample digestedwith restriction enzyme Hind III. Also, an about 5.0 kbp band wasobserved in the sample digested with restriction enzyme Nsp V. For othersamples, any amplification was not observed in this reaction.

Then, with 1 μl of the primary PCR reaction solution, obtained from thesample digested with restriction enzyme Hind III, used as the template,nested PCR using TAKARA LA PCR Kit (TAKARA SHUZO Co., Ltd.) was carriedout by using primer KOM-2 (SEQ ID NO 59) as a sense primer and primerKOM-5 (SEQ ID NO 60) as an antisense primer in the reaction system witha total volume of 50 μl. The reaction was carried out by repeating acycle of 94° C. (1 minute), 98° C. (20 seconds), and 67° C. (10 minutes)30 times, finally followed by 72° C. (10 minutes). After the reaction, 5μl of the reaction solution underwent 1% agarose gel electrophoresis,indicating that only an about 3.3 kbp band was observed. Then, theresulting DNA fragment was recovered from the gel and subjected toend-blunting using DNA Blunting Kit (TAKARA SHUZO Co., Ltd.),phosphorylation of the PCR product using the 5'-Terminal-Labeling KitMEGALABEL™ (TAKARA SHUZO Co., Ltd.) at the 5'-terminus, and thentransfer into the Hinc II site of pUC119 (TAKARA SHUZO Co., Ltd.). Theresulting plasmid was transformed into E. coli JM109.

Of the obtained colonies, 15 colonies were screened for plasmidscontaining inserted fragments of appropriate lengths by colony-pickingPCR using M13 Primer M4 (TAKARA SHUZO Co., Ltd.) and M13 Primer RV(TAKARA SHUZO Co., Ltd.), indicating that 8 out of 15 colonies werepositive. Of them, plasmids were extracted from these 6 colonies andnamed as pKOM-1, pKOM-2, pKOM-3, pKOM-4, pKOM-5, and pKOM-6,respectively.

Each of pKOM-1, pKOM-2, pKOM-3, pKOM-4, pKOM-5, and pKOM-6 was subjectedto the sequence analysis of the both termini of respective, insertedfragments according to the Sanger method using M13 Primer M4 (TAKARASHUZO Co., Ltd.) and M13 Primer RV (TAKARA SHUZO Co., Ltd.). Comparisonof these sequences with the sequence [EP-0562836 A1 (1993)] of theoriginal wheat EXT cDNA revealed that the overlapping portions werecompletely identical. However, 2 to 3 base substitution was detectedwithin the sequenced range. It was conceived as in the case of tomatothat this substitution involved a mistake induced by the nested PCR inthe polymerase reaction.

FIG. 16 illustrates the restriction map of the about 3.3 kbp insertedfragment amplified with primers KOM-2 and KOM-5. In the figure, theupper part in the restriction map corresponds to the nucleotide sequenceof primer KOM-5 and the lower part corresponds to the nucleotidesequence of primer KOM-2.

Complete digestion of pKOM-1, pKOM-2, pKOM-3, pKOM-4, pKOM-5, and pKOM-6with restriction enzyme Hind III, followed by agarose electrophoresis,revealed formation of two bands at about 4.2 kpb and at about 2.0 kbpfor pKOM-1, pKOM-3, and pKOM-5 as well as two bands at about 4.9 kbp andat about 1.3 kbp for pKOM-2, pKOM-4, and pKOM-6. This observationindicated that the EXT was inserted in the reverse directions for thethree bands of pKOM-1, pKOM-3, and pKOM-5 and for the three bands ofpKOM-2, pKOM-4, and pKOM-6. In addition, it was revealed from theresults on PCR, which was carried out, with these plasmids used astemplates, by using primer KOM-2 (SEQ ID NO 59) and M13 Primer M4(TAKARA SHUZO Co., Ltd.) or M13 Primer RV (TAKARA SHUZO Co., Ltd.), thatthe about 1.3-kbp DNA fragment contained the wheat EXT gene promoterregion, among the DNA fragments of about 2.0 kbp and about 1.3 kbp whichwere separated upon complete digestion of the inserted fragment withrestriction enzyme Hind III.

Then, pKOM-1 was completely digested with restriction enzyme Hind IIIand the about 1.3-kbp DNA fragment, namely a DNA fragment containing thewheat EXT gene promoter region, was subjected to purification by agaroseelectrophoresis, followed by self-ligation using TAKARA DNA Ligation Kit(TAKARA SHUZO Co., Ltd.). The resulting plasmids were transformed intoE. coli JM109.

Of the obtained colonies, 6 colonies were examined for the size of theinserted fragment by PCR to detect an about 1.3 kbp DNA fragment. Then,plasmids were extracted from 3 colonies and named as pKEP-1, pKEP-2, andpKEP-3, respectively. Complete digestion of these pKEP-1, pKEP-2, andpKEP-3 with restriction enzymes EcoR I, Sac I, Kpn I, Sma I, BamH I, XbaI, Pst I, and Hind III, followed by agarose electrophoresis, was carriedout to prepare their restriction maps. Of them, the restriction map ofpKEP-1 is shown in FIG. 17.

Next, each of KEP-1, KEP-2, and KEP-3 was completely digested withrestriction enzyme Sac I and the about 3.8 kbp band was subjected topurification by agarose electrophoresis, followed by self-ligation usingTAKARA DNA Ligation Kit (TAKARA SHUZO Co., Ltd.). The resulting plasmidswere named as pKEPS-1, pKEPS-2, and pKEPS-3.

Furthermore, each of pKEPS-1, pKEPS-2, and pKEPS-3 was subjected todouble digestion with restriction enzyme EcoR I-Pst I and purificationof the about 1.1 kbp band by agarose electrophoresis. Then, this DNAfragment was ligated to the molecule obtained by double digestion ofpUC19 (TAKARA SHUZO Co., Ltd.) with restriction enzymes EcoR I-Pst I.The resulting plasmids were transformed into E. coli JM109.

Of the obtained colonies, 5 colonies were screened for the size of theinserted fragment of the about 1.1 kbp by PCR using M13 Primer M4(TAKARA SHUZO Co., Ltd.) and M13 Primer RV (TAKARA SHUZO Co., Ltd.). Theplasmids were extracted from positive colonies and named as pKEPEP-1,KEPPEP-2, and KEPPEP-3, respectively.

Each of these pKEP-1, pKEP-2, pKEP-3, pKEPS-1, pKEPS-2, pKEPS-3,pKEPEP-1, KEPPEP-2, and KEPPEP-3 was subjected to the sequence analysisof the nucleotide sequence of the respective, inserted fragment portionaccording to the Sanger method using M13 Primer M4 (TAKARA SHUZO Co.,Ltd.) and M13 Primer RV (TAKARA SHUZO Co., Ltd.).

On the basis of comprehensive interpretation of there results, theentire nucleotide sequence in the promoter region upstream from thiswheat EXT N-terminal amino acid sequence contained in pKEP-1 wasdetermined. This sequence is shown by SEQ ID NO 8 in the SequenceListing.

EXAMPLE 19

Analysis of Expression Mode for Tomato EXT Gene

(1) Preparation of Total RNA

Each of 5 g tissues collected from leaves, stems (during elongation andafter elongation), and fruits [mature green fruit (of which the surfaceis green and a gelly substance is formed in the inside) and mature redfruit] of Arisa Craig, a tomato plant, was frozen, and pulverized usinga mortar in liquid nitrogen. The pulverized tissues were mixed with 5 mlof an extraction solution [0.2 M Tris-HCl (pH: 9.0), 0.1 M NaCl, 10 mMEDTA, 0.5% SDS, and 14 mM 2-mercaptoethanol], and 5 ml of aphenol-chloroform-isoamyl alcohol (50:49:1) mixture and stirredvigorously. The resulting suspension was centrifuged to separate anaqueous layer. This procedure was repeated twice. The separated aqueouslayer was mixed with a 1/10 volume of 3 M sodium acetate, cooled withice for 20 minutes, and centrifuged. The supernatant was recovered,mixed with ethanol, and then centrifuged to obtain a precipitate. Thisprecipitate was dissolved in 2 ml of a TE/HPRI solution [10 mM Tris-HCl,1 mM EDTA, 5U/ml Rnase inhibitor (TAKARA SHUZO Co., Ltd.) and 1 mMdithiothreitol (DTT)], mixed with a 1/4 volume of 10 M lithium chloride,cooled with ice for 2 hours, and then centrifuged. The obtainedprecipitate was dissolved in 0.5 ml of the TE/HPR I solution, mixed with3 M sodium acetate and ethanol, and then centrifuged to obtain an RNAprecipitate.

(2) Northern Hybridization

Each of a fragment of the tomato EXT cDNA [EP-0562836 A1 (1993)] and acDNA fragment of tomato fruit polygalacturonase (Tomato PG) [Molecular &General Genetics, 208, 30-36 (1987)] was labeled with [α-³² P]dCTP usingBcaBEST™ Labeling Kit (TAKARA SHUZO Co., Ltd.) to prepare a probe fornorthern hybridization, respectively. The northern hybridization wascarried out in the following way according to a modification of themethod described in "Molecular Cloning, A laboratory Manual", SecondEdition, Chapter 7, pp. 7.39-7.52 (T. Maniatis et al., Published by ColdSpring Harbor Laboratory Press in 1989). That is to say, 2 μg of theextracted RNA was subjected to electrophoresis with formaldehyde-runningagarose gel (1%), followed by northern blotting on a nylon membrane(Hybond-N⁺) overnight. After RNA was immobilized by irradiation with aultraviolet transilluminator (254 nm) for 3 minutes, the membrane wassubjected to pre-hybridization in 25 ml of a pre-hybridization buffersolution (6×SSC, 0.1% SDS, 5×Denhardt's solution, and 100 μg/ml salmonsperm DNA) at 65° C. for 2 hours.

The ³² P-labeled probe prepared by the above-mentioned method was addedto 25 ml of a pre-hybridization buffer solution (6×SSC, 0.1% SDS,5×Denhardt's solution). To this probe solution was added the membraneobtained by the pre-hybridization and hybridization was carried out at65° C. overnight.

After the hybridization, the membrane was washed thrice with a washingsolution containing 2×SSC and 0.1% SDS at 65° C. for 20 minutes. Afterbeing dried, the membrane was exposed overnight at -80° C. in a cassettein which an X-ray film (Kodak) was placed to prepare an autoradiograph.

The results are shown in FIG. 18. That is to say, FIG. 18 illustratesthe northern hybridization using the tomato tissues, wherein theexpression of a tomato EXT mRNA was shown in the upper row, theexpression of a Tomato PG mRNA was shown in the middle row, and the rRNAlevels were shown in the lower row. Also in the figure, lane 1 indicatesthe mature red fruit, lane 2 the mature green fruit (of which thesurface is green and a gelly substance is formed in the inside), lane 3the elongating stems, lane 4 the elongated stems, and lane 5 the leaves.

As can be seen from FIG. 18, it was revealed on comparison of theexpression level of the tomato EXT mRNA between each of the planttissues that an intense expression was observed particularly in themature green fruit and in the elongating stems. In contrast, the TomatoPG mRNA used as a control, on comparison of the expression level betweeneach of the plant tissues, was expressed intensely in the mature redfruit.

(3) RT-PCR Using Tomato Fruits

According to the procedure as shown in Example 19 (1), total RNAs wereprepared from 10 kinds of fruits in different ripening stages rangingfrom an immature green fruit (of which the surface is green but a gellysubstance is not formed in the inside) to a mature red fruit of ArisaCraig, a tomato plant. One μg each of these total RNA was utilized forRT-PCR using TAKARA RNA PCR Kit with AMV Version 2 (TAKARA SHUZO Co.,Ltd.) in the following manner to analyze the expression of the tomatoEXT mRNA and Tomato PG mRNA.

The reverse transcription reaction was carried out by using a randomprimer (9 mer) attached in the kit at 30° C. (1 minute), 55° C. (15minutes), 99° C. (5 minutes) and 5° C. (5 minutes). Then, with the wholereaction solution used as the template, the PCR reaction was carried outusing combinations of:

1) primer TOM-1 (SEQ ID NO 61) and primer TOM-2 (SEQ ID NO 62),synthesized on the basis of the tomato EXT cDNA fragment [EP-0562836 A1(1993)], and

2) primer PG-SP3 (SEQ ID NO 63) and primer PG-AP2 (SEQ ID NO 64),synthesized on the basis of Tomato PG cDNA fragment [Molecular & GeneralGenetics, 208, 30-36 (1987)]. The reaction was carried out by repeating25 times a cycle at 94° C. (0.5 minute), 55° C. (1 minute), and 72° C.(1 minute). After the reaction, an aliquot of the reaction solution wassubjected to 1% agarose gel electrophoresis. The results are shown inFIG. 19. That is to say, FIG. 19 illustrates the RT-PCR using the tomatotissues, wherein the expression of the tomato EXT, amplified by theprimers described in 1) mentioned above, in each of the ripening stageswas shown in the upper row and the expression of Tomato PG (theamplification product), amplified by the primers described in 2)mentioned above, in each of the ripening stages was shown in the lowerrow. Also in the figure, each lane with increasing the number indicatesthe increasing ripening stages for the fruit. In other words, lanes 1and 2 correspond to the immature green fruit (of which the surface isgreen but a gelly substance is not formed in the inside), lanes 3 and 4to the mature green fruit (of which the surface is green and a gellysubstance is formed in the inside), lanes 5 and 6 to a turning fruit (10to 30% of the fruit surface turns red), lanes 7 and 8 to a pink fruit(30 to 60% of the fruit surface turns red), and lanes 9 and 10 to themature red fruit (100% of the fruit surface turns red).

As can be seen from FIG. 19, it was revealed on comparison of theexpression of the tomato EXT at each ripening stages that an intenseexpression of the tomato EXT was induced at the immature green to maturegreen stages, as the amplification product (about 913 bp) was detectedin lane 1 to lane 4 corresponding to these stages. On the other hand, itwas revealed that the Tomato PG mRNA used as a control was expressedintensely in the turning and pink stages corresponding to lanes 5 to 9where the amplification product (about 561 bp) was detected.

These results revealed that the tomato EXT promoter was a promoter thatinduces an intense gene expression particularly in growing stems andenlarging fruits (immature to mature green). That is to say, it wasrevealed that the gene expression was induced in each case at the siterequired for the reconstitution of plant cell wall xyloglucan and at thestage required for the reconstitution of plant cell wall xyloglucan.

EXAMPLE 20

Transient Assay Using Tobacco Culture Cells

(1) Construction of Plasmids for Transfer

First, construction of respective plasmids for the transfer wasperformed in order to transfer a plasmid containing a chimeric gene of apromoter region and the GUS gene into the protoplasts of tobacco BY2culture cells by using the electroporation method.

1. Preparation of Plasmid Containing Chimeric Gene of DNA FragmentContaining Azuki Bean EXT2 Gene Promoter Region and the GUS Gene(Transcriptional Fusion)

A plasmid containing a chimeric gene of a DNA fragment containing theazuki bean EXT2 gene promoter region and the GUS gene was constructed asillustrated in FIG. 20. That is to say, pBI221 (Clontech) having thecauliflower mosaic virus 35S promoter, the E. coli-origin GUS gene, anda transcription termination sequence cassette originating from nopalinesynthetase was utilized.

First, in order to remove the cauliflower mosaic virus 35S promoterregion in pBI221, this plasmid was subjected to digestion withrestriction enzymes Hind III and Sma I (TAKARA SHUZO Co., Ltd.), andthen purification of the objective fragment other than the 35S promoterregion by agarose gel electrophoresis followed by cutting-off. Next,pVX2P501 prepared in Example 13 was subjected to complete digestion withrestriction enzymes EcoR I and Hinc II, and then purification of theabout 0.5 kbp inserted fragment by agarose gel electrophoresis followedby cutting-off. Also, pEXT2pro(F) f3 prepared in Example 13 wassubjected to complete digestion with restriction enzymes Hind III andEcoR I, and then purification of the about 2.55 kbp inserted fragment byagarose gel electrophoresis followed by cutting-off. These DNA fragmentswere ligated together and then transformed into E. coli JM 109 strain.This plasmid was named as pVAEXT2GUS and E. coli JM 109 straintransformed with pVAEXT2GUS was named as Escherichia coli JM109/pVAEXT2GUS. This pVAEXT2GUS formed an about 3.4 kbp band bydigestion with restriction enzymes Hind III and SnaB I, followed byagarose gel electrophoresis, thereby revealing that this plasmidcontained the full length of the about 3.0 kbp azuki bean EXT2 genepromoter region.

2. Preparation of Plasmid Containing Chimeric Gene of DNA FragmentContaining Azuki Bean EXT3 Gene Promoter Region and the GUS Gene(Transcriptional Fusion)

A vector containing a chimeric gene of a DNA fragment containing theazuki bean EXT3 gene promoter region and the GUS gene was constructed asillustrated in FIG. 21. That is to say, pBI221 (Clontech) having thecauliflower mosaic virus 35S promoter, the E. coli-origin GUS gene, anda transcription termination sequence cassette originating from nopalinesynthetase was utilized.

First, in order to remove the cauliflower mosaic virus 35S promoterregion in pBI221, this plasmid was subjected to digestion withrestriction enzymes Hind III and Xba I, and then purification of theobjective fragment other than the 35S promoter region by agarose gelelectrophoresis followed by cutting-off. Next, with about 0.3 μg ofpVX3P206 prepared in Example 14 used as the template, PCR was carriedout by using primer VX3UH (SEQ ID NO 65), which situated in a regiondownstream from Nsp V in the azuki bean EXT3 gene promoter region inpVX3P206, and primer VX3LX (SEQ ID NO 66), the sequence just before thetranslation initiation point. These primer VX3UH (SEQ ID NO 65) andprimer VX3LX (SEQ ID NO 66) were synthesized so that the Xba I site andHind III site were transferred into the both termini of the PCR product,respectively. The reaction was carried out by repeating a cycle of 94°C. (1 minute), 55° C. (1 minute), and 72° C. (2 minutes) 10 times. Afterthe reaction, 5 μl of the reaction solution underwent 1% agarose gelelectrophoresis to detect an about 0.4 kbp band in addition to thetemplate plasmid band. Since the Xba I site and Hind III site had beentransferred into primer VX3UH (SEQ ID NO 65) and primer VX3LX (SEQ ID NO66), respectively, this about 0.4 kbp DNA fragment was subjected topurification by agarose gel electrophoresis, digestion with restrictionenzymes Hind III and Xba I, ligation with the previously-purified pBI221Hind III-Xba I DNA fragment, and then transformation into E. coli JM 109strain. This plasmid was named as pVAEXT3GUS and E. coli JM 109 straintransformed with pVAEXT3GUS was named as Escherichia. coli JM109/pVAEXT3GUS.

This pVAEXT3GUS formed an about 0.4 kbp band by digestion withrestriction enzymes Hind III and Xba I, followed by agarose gelelectrophoresis, thereby revealing that this plasmid contained the fulllength of the about 0.4 kbp azuki bean EXT3 gene promoter region.

3. Preparation of Plasmid Containing Chimeric Gene of DNA FragmentContaining Azuki Bean XRP1 Gene Promoter Region and the GUS Gene(Translational Fusion)

A vector containing a translational fusion chimeric gene of a DNAfragment containing the azuki bean XRP1 gene promoter region and the GUSgene was constructed as illustrated in FIG. 22. That is to say, pBI221(Clontech) having the cauliflower mosaic virus 35S promoter, the E.coli-origin GUS gene, and a transcription termination sequence cassetteoriginating from nopaline synthetase was utilized.

First, pBI221 was subjected to digestion with restriction enzymes Xba Iand Sma I, and then purification of the objective DNA fragment byagarose gel electrophoresis followed by cutting-off. Next, pXRG302prepared in Example 15 was subjected to double digestion withrestriction enzymes Xba I-Hinc II and then purification of the about 1.1kbp inserted fragment by agarose gel electrophoresis followed bycutting-off. This DNA fragment was ligated to the previously-purifiedpBI221 DNA fragment and then transformed into E. coli JM 109 strain.

A plasmid was prepared from the colonies obtained. Next, in order toremove the cauliflower mosaic virus 35S promoter region in this plasmid,the plasmid was subjected to digestion with restriction enzymes Hind IIIand Xba I, self-ligation, and then transformation into E. coli JM 109strain. A plasmid was purified from the colonies obtained. This plasmidwas named as pVAXRP1tlGUS and E. coli JM 109 strain transformed withpVAXRP1tlGUS was named as Escherichia. coli JM 109/pVAXRP1tlGUS. PCR,which was carried out with this pVAXRP1tlGUS used as a template and byusing M13 Primer M4 (TAKARA SHUZO Co., Ltd.) and M13 Primer RV (TAKARASHUZO Co., Ltd.), followed by agarose gel electrophoresis, resulted information of an about 3.0 kbp band, thereby revealing that this plasmidcontained the full length of the about 1.1 kbp azuki bean XRP1 genepromoter region.

Furthermore, when the nucleotide sequence of a portion upstream from theGUS-gene in pVAXRP1tlGUS up to the promoter region determined, it wasconfirmed that the gene originating from pBI221 (SEQ ID NO 68) wasintegrated after a gene encoding the azuki bean XRP1 N-terminal aminoacid sequence in such a manner that GUS could be expressed so as to formthe translational-fusion protein of GUS having the azuki bean XRP1N-terminal amino acid sequence by this pVAXRP1tlGUS.

4. Preparation of Plasmid Containing Chimeric Gene of DNA FragmentContaining Tomato EXT Gene Promoter Region and the GUS Gene(Transcriptional Fusion)

A vector containing a chimeric gene of a DNA fragment containing thetomato EXT gene promoter region (about 1.4 kbp) and the GUS gene wasconstructed as illustrated in FIG. 23. That is to say, pBI221 (Clontech)having the cauliflower mosaic virus 35S promoter, the E. coli-origin GUSgene, and a transcription termination sequence cassette originating fromnopaline synthetase was utilized.

First, in order to remove the cauliflower mosaic virus 35S promoterregion in pBI221, this plasmid was subjected to double digestion withrestriction enzymes Hind III-Xba I and then purification of theobjective fragment other than the 35S promoter region by agarose gelelectrophoresis followed by cutting-off. Next, with about 0.3 μg ofpLXG103 used as the template, PCR was carried out by using primer LXUH1(SEQ ID NO 69), which situated in a region downstream from Hind III sitein the tomato EXT gene promoter region in pLXG103 prepared in Example16, and primer LXLX (SEQ ID NO 70), the sequence just before thetranslation startpoint. These primer LXUH1 (SEQ ID NO 69) and primerLXLX (SEQ ID NO 70) were synthesized so that the Hind III site and Xba Isite were added and transferred into the termini of the PCR product,respectively. The reaction was carried out by repeating a cycle of 94°C. (1 minute), 55° C. (1 minute), and 72° C. (2 minutes) 10 times. Afterthe reaction, 5 μl of the reaction solution underwent 1% agarose gelelectrophoresis to detect an about 1.4 kbp band in addition to thetemplate plasmid band. Since the Hind III site and Xba I site had beentransferred into primer LXUH1 (SEQ ID NO 69) and primer LXLX (SEQ ID NO70), respectively, this about 1.4 kbp DNA fragment was subjected topurification by agarose gel electrophoresis, digestion with restrictionenzymes Hind III and Xba I, ligation with the previously-purified pBI221DNA fragment, and then transformation into E. coliJM 109 strain. Thisplasmid was named as pLEEXT1.4GUS and E. coli JM 109 strain transformedwith pLEEXT1.4GUS was named as Escherichia. coli JM 109/pLEEXT1.4GUS.

This pLEEXT1.4GUS formed an about 1.4 kbp band by digestion withrestriction enzymes Hind III and Xba I, followed by agarose gelelectrophoresis, thereby revealing that this plasmid contained the about1.4 kbp tomato EXT3 gene promoter region.

Moreover, as shown in FIG. 24, a plasmid having a fusion gene(transcriptional fusion) with the GUS gene using only a regionhomologous with the tobacco EXT gene promoter region was prepared.

With about 0.3 μg of pLXG103 prepared in Example 16 used as thetemplate, PCR was carried out by using primer LXUH2 (SEQ ID NO 71),which situated in the 5'-downstream from a region homologous with thetobacco EXT gene promoter region and the tomato EXT gene promoter regionin pLXG103, and primer LXLX (SEQ ID NO 70), the sequence just before thetranslation startpoint. These primer LXUH2 (SEQ ID NO 71) and primerLXLX (SEQ ID NO 70) were synthesized so that the Hind III site and Xba Isite were added and transferred into the both termini of the PCRproduct, respectively. The reaction was carried out by repeating a cycleof 94° C. (1 minute), 55° C. (1 minute), and 72° C. (2 minutes) 10times. After the reaction, 5 μl of the reaction solution underwent 1%agarose gel electrophoresis to detect an about 0.7 kbp band in additionto the template plasmid band. Since the Hind III site and Xba I site hadbeen transferred into primer LXUH2 (SEQ ID NO 71) and primer LXLX (SEQID NO 70), respectively, this about 0.7 kbp DNA fragment was subjectedto purification by agarose gel electrophoresis, digestion withrestriction enzymes Hind III and Xba I, ligation with thepreviously-purified pBI221 DNA fragment, and then transformation into E.coli JM 109 strain. This plasmid was named as pLEEXT0.7GUS and E. coliJM 109 strain transformed with pLEEXT0.7GUS was named as Escherichia.coli JM 109/pLEEXT0.7GUS.

This pLEEXT0.7GUS formed an about 0.7 kbp band by digestion withrestriction enzymes Hind III and Xba I, followed by agarose gelelectrophoresis, thereby revealing that this plasmid contained the about0.7 kbp tomato EXT gene promoter region.

5. Preparation of Plasmid Containing Chimeric Gene of DNA FragmentContaining Tomato XRP Gene Promoter Region and the GUS Gene(Translational Fusion)

A vector containing a gene (SEQ ID NO 72) encoding the tomato EXTN-terminal amino acid sequence and a translational-fusion of an about4.9 kbp DNA fragment with the GUS gene was constructed as illustrated inFIG. 25. That is to say, pBI221 (Clontech) having the cauliflower mosaicvirus 35S promoter, the E. coli-origin GUS gene, and a transcriptiontermination sequence cassette originating from nopaline synthetase wasutilized.

First, in order to remove the cauliflower mosaic virus 35S promoterregion in pBI221, this plasmid was subjected to digestion withrestriction enzymes Pst I and BamH I, and then purification of theobjective DNA fragment other than the 35S promoter region by agarose gelelectrophoresis followed by cutting-off. Next, pLXG601 prepared inExample 16 was subjected to digestion with restriction enzymes Pst I andBamH I, and then purification of the about 4.9 kbp inserted fragment byagarose gel electrophoresis followed by cutting-off. This DNA fragmentwas ligated to the previously-purified pBI221 DNA fragment and thentransformed into E. coli JM 109 strain. This plasmid was named aspLEEXTtl4.9GUS and E. coli JM 109 strain transformed with pLEEXTtl4.9GUSwas named as Escherichia. coli JM 109/pLEEXTtl4.9GUS. Complete digestionof this pLEEXTtl4.9GUS with restriction enzymes Pst I and BamH I,followed by agarose gel electrophoresis, resulted in formation of anabout 4.9 kbp band, thereby revealing that this plasmid contained theabout 4.9 kbp tomato EXT gene promoter region.

Furthermore, when the nucleotide sequence of a portion upstream from theGUS gene up to the promoter region in pLEEXTtl4.9GUS, it was confirmedthat a gene originating from pBI221 (SEQ ID NO 73) was integrated aftera gene encoding the tomato EXT N-terminal amino acid sequence in such amanner that GUS could be expressed so as to form thetranslational-fusion protein of GUS having the tomato EXT N-terminalamino acid sequence by this pLEEXTtl4.9GUS.

Next, a vector containing a translational fusion of an about 1.4 kbp DNAfragment containing a gene (SEQ ID NO 72) encoding the tomato EXTN-terminal amino acid sequence and a promoter region with the GUS genewas constructed as illustrated in FIG. 26. That is to say, pBI221(Clontech) having the cauliflower mosaic virus 35S promoter, the E.coli-origin GUS gene, and a transcription-termination sequence cassetteoriginating from nopaline synthetase was utilized.

First, in order to remove the cauliflower mosaic virus 35S promoterregion in pBI221, this plasmid was subjected to digestion withrestriction enzymes Hind III and BamH I, and then purification of theobjective DNA fragment other than the 35S promoter region by agarose gelelectrophoresis followed by cutting-off. Next, pLXP101 prepared inExample 16 was subjected to digestion with restriction enzymes Hind IIIand BamH I, and then purification of the about 1.4 kbp inserted fragmentby agarose gel electrophoresis followed by cutting-off. This DNAfragment was ligated to the previously-purified pBI221 DNA fragment andthen transformed into E. coli JM 109 strain. This plasmid was named aspLEEXTtl1.4GUS and E. coli JM 109 strain transformed with pLEEXTtl1.4GUSwas named as Escherichia. coli JM 109/pLEEXTtl1.4GUS.

Complete digestion of this pLEEXTtl1.4GUS with restriction enzymes HindIII and BamH I, followed by agarose gel electrophoresis, resulted information of an about 1.4 kbp band, thereby revealing that this plasmidcontained the about 1.4 kbp tomato EXT gene promoter region.

Furthermore, when the nucleotide sequence of a portion upstream from theGUS gene up to the promoter region in pLEEXTtl1.4GUS was determined, itwas confirmed that a gene originating from pBI221 (Sequence No. 73) wasintegrated after a gene encoding the tomato EXT N-terminal amino acidsequence is such a manner that GUS could be expressed so as to form thetranslational fusion protein of GUS having the tomato EXT N-terminalamino acid sequence by this pLEEXTtl1.4GUS.

6. Preparation of Plasmid Containing Chimeric Gene of DNA FragmentContaining Tobacco EXT Gene Promoter Region and the GUS Gene(Transcriptional Fusion)

A vector containing a chimeric gene of a DNA fragment containing thetobacco EXT gene promoter region and the GUS gene was constructed asillustrated in FIG. 27. That is to say, pBI221 (Clontech) having thecauliflower mosaic virus 35S promoter, the E. coli-origin GUS gene, anda transcription termination sequence cassette originating from nopalinesynthetase was utilized.

First, in order to remove the cauliflower mosaic virus 35S promoterregion in pBI221, this plasmid was subjected to digestion withrestriction enzymes Pst I and Xba I, and then purification of theobjective fragment other than the 35S promoter region by agarose gelelectrophoresis followed by cutting-off. Next, with about 0.3 μg ofpNXG102 prepared in Example 17 used as the template, PCR was carried outby using primer NXUP (SEQ ID NO 74), which situated in a regiondownstream from Hind III site in the tobacco EXT gene promoter region inpNXG102, and primer NXLX (SEQ ID NO 75), the sequence just before thetranslation initiation point. These primer NXUP (SEQ ID NO 74) andprimer NXLX (SEQ ID NO 75) were synthesized so that the Pst I site andXba I site were added and transferred into the both termini of the PCRproduct, respectively. The reaction was carried out by repeating a cycleof 94° C. (1 minute), 55° C. (1 minute), and 72° C. (2 minutes) 10times. After the reaction, 5 μl of the reaction solution underwent 1%agarose gel electrophoresis to detect an about 0.8 kbp band in additionto the template plasmid band. Since the Pst I site and Xba I site hadbeen transferred into primer NXUP (SEQ ID NO 74) and primer NXLX (SEQ IDNO 75), respectively, this about 0.8 kbp DNA fragment was subjected topurification by agarose gel electrophoresis, digestion with restrictionenzymes Pst I and Xba I, ligation with the previously-purified pBI221DNA fragment, and then transformation into E. coli JM 109 strain. Thisplasmid was named as pLEEXT0.8GUS and E. coli JM 109 strain transformedwith pLEEXT0.8GUS was named as Escherichia. coli JM 109/pLEEXT0.8GUS.

This pLEEXT0.8GUS formed an about 0.8 kbp band by digestion withrestriction enzymes Pst I and Xba I, followed by agarose gelelectrophoresis, thereby revealing that this plasmid contained the about0.8 kbp tobacco EXT gene promoter region.

7. Preparation of Plasmid Containing Chimeric Gene of DNA FragmentContaining Wheat EXT Gene Promoter Region and the GUS Gene(Transcriptional Fusion)

A vector containing a chimeric gene of a DNA fragment containing thewheat EXT gene promoter region and the GUS gene was constructed asillustrated in FIG. 28. That is to say, pBI221 (Clontech) having thecauliflower mosaic virus 35S promoter, the E. coli-origin GUS gene, anda transcription termination sequence cassette originating from nopalinesynthetase was utilized.

First, in order to remove the cauliflower mosaic virus 35S promoterregion in pBI221, this plasmid was subjected to digestion withrestriction enzymes Hind III and Sma I, and then purification of theobjective DNA fragment other than the 35S promoter region by agarose gelelectrophoresis followed by cutting-off. Next, about 2 μg of pKEP-1prepared in Example 18 was subjected to complete digestion withrestriction enzymes Hind III and Nae I, and then purification of theabout 0.6 kbp and about 0.5-kbp DNA fragments by agarose gelelectrophoresis followed by cutting-off.

Both of the DNA about 0.6-kbp and about 0.5-kbp fragments were ligatedto the previously-purified pBI221 DNA fragment and then transformed intoE. coli JM 109 strain. This plasmid was named as pTAEXT1.1GUS and E.coli JM 109 strain transformed with said plasmid was named asEscherichia. coli JM 109/pTAEXT1.1GUS.

Digestion of this pTAEXT1.1GUS with restriction enzymes Hind III andEcoR I, followed by agarose gel electrophoresis, resulted in formationof an about 3.3 kbp band, thereby revealing that this plasmid containedthe about 1.1 kbp wheat EXT gene promoter region.

(2) Gene Transfer by Electroporation

In order to transfer each of plasmids, prepared in Example 20-1 to 20-7as described above, into tobacco BY2 culture cells by theelectroporation method, the tobacco BY2 culture cells were treated withan enzyme solution (pH: 5.5) containing 1% cellulase-ONOZUKA (YakultHonsha Co., Ltd.), 0.1% pectolyase Y23 (SEISHIN Corporation), and 0.4 Mmannitol at 30° C. for 2 hours to be converted into cell wall-freeprotoplasts. A suspension of the 2×10⁶ protoplasts of the tobacco BY2culture cells in an electroporation buffer solution (70 mM KCl, 5 mMYES, and 0.3 M mannitol, pH 5.8) was mixed with 3 pmol of each ofplasmids, prepared in paragraphs 1 to 7 as described above, and a 10%PEG6000/electroporation buffer solution with stirring. An electric pulse(300 V, 125 μF) using Gene Pulser II (Bio-Rad Laboratories) was appliedto the resulting mixture to transfer the DNA into the plant cells.

The cells were incubated in the Linsmaier-Skoog culture medium[Physiologia Plantarum, 18, 100 (1965)] containing 0.2 mg/l 2,4-D as anauxin, 1% sucrose, and 0.4 M. mannitol at 26° C. for 40 hours after thetransfer. The cells were recovered and a mixture of the recovered cellsin 200 μl of an extraction buffer solution [50 mM phosphate buffer (pH7.0), 10 mM EDTA, 0.1% Triton X-100, 0.1% Sarkosyl, and 10 mM2-mercaptoethanol] placed in an Eppendorf tube was subjected toultra-sonication on ice for 30 seconds by using a ultrasonicator W-225(Heatsystems-Ultrasonics) with setting the output control at 1.5 and theduty cycle at 50%. Then, a supernatant isolated by centrifugation wasused for the assay of the GUS activity and the assay of the amount ofprotein.

(3) Measurement of Promoter Activity The reaction was carried out byadding 45 μl of the extraction buffer solution and 25 μl of a 4 mM 4-MUGsubstrate to each 30 μl of the above-mentioned extracts placed in a96-well microtiter plate for fluorescence. After 5, 35, and 95 minutes,the reaction was terminated by addition of 50 μl of areaction-termination solution (1 M Na₂ CO₃). Then, the specificfluorescence emitted by 4-MU, the reaction product, at an excitationwavelength of 365 nm and fluorescence wavelength of 455 nm, was measuredwith a fluorescence plate reader [Fluoroscan II (Labosystems)].

Moreover, the protein quantity was assayed by a procedure exemplified asfollows. Thus, 2, 5, 10, 15, 20, and 30 μl of a 1/5-diluted solution ofthe extract or an 800 μg/ml BSA standard solution (20 μl of the extractbuffer solution is mixed with 80 μl of 1 mg/ml BSA) were placed in a96-well microtiter plate and thereto were added respectively 158, 155,150, 145, 140, and 130 μl of distilled water and 40 μl of the assayreagent in Bio-Rad Protein Assay Kit (Bio-Rad Laboratories). After beingstirred slowly and then allowed to stand for 20 minutes at roomtemperature, the mixture was measured by a plate reader (wavelength: 590nm) within 60 minutes to assay the protein quantity.

The GUS activity was measured in the following way. At the same timewhen the above assays were carried. out, the fluorescence intensities ofthe 4-MU standard solutions were measured and the results were plottedon a graph with the 4-MU quantity (pmol) at the x-axis and thefluorescence intensity at the y-axis. Then, the 4-MU quantity per onefluorescence unit was obtained from the slope and, further, the resultson the samples were plotted on a graph with the time (minute) at thehorizontal axis and the fluorescence intensity at the vertical axis toobtain the increasing rate of the fluorescence intensity and then toobtain the decomposition rate of 4-MUG equal to the GUS activity. Inaddition, the GUS specific activity was obtained from the amount ofprotein. The results are shown in FIG. 29. In other words, FIG. 29illustrates comparison of the GUS-specific activity of the transformedtobacco BY2 culture cells, wherein the specific activity value upon thetransfer of pLEEXT1.4GUS is taken as 100 for obtaining the GUS-specificactivity upon the transfer of each plasmid and plotting each promoteractivity on a graph, thereby enabling comparison the transferexperiments carried out 7 times in total.

In the figure, the GUS-specific activity values upon the transfer ofeach plasmid are indicated at the horizontal axis, with the specificactivity value upon the transfer of pLEEXT1.4GUS being taken as 100, andthe plasmids used in the experiments are indicated at the vertical axis.The n numbers are 2 to 7.

From these results, it was confirmed that the DNA fragment containingthese EXT gene promoter regions exhibited an activity more intense thanthat of the cauliflower mosaic virus 35S promoter that had been said tobe expressed intensely in the plants. As can be seen from this figure,it could be revealed that, particularly, the activities of the azukibean XRP1 and tomato EXT promoters were extremely high and that theefficiency was better by the translational fusion from comparison of thetomato EXT promoters.

As described hereinabove, the present invention provides prompters ofgenes and family genes thereof encoding the endo-xyloglucan transferase(EXT) to be expressed in a specific manner at the site and stagerequired for the reconstitution of plant cell wall xyloglucan. Moreover,the present invention provides methods for cloning the promoters of theEXT genes and family genes thereof, plant transformation vectorscontaining the promoters of the EXT genes and family genes thereof aswell as methods for preparing them, methods for regulating theexpression of the promoters of the EXT genes and family genes thereof,and methods for controlling the plant morphology and plants using thepromoters of the EXT genes and family genes thereof.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                  - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 75                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1875 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - AAGCTTTTTG CACATATTTG CAGCAGTAGA CAATGCCACT CGCTGAAAAA TA -             #TGATCTCC     60                                                                 - - CAGAATTTTG GCACAAAAAA TATATCCTAA CTAATATTTG ACTCTATCTA AG -            #ATACCACC    120                                                                 - - TGACATCAAA TGTTTCAATT TTATAGTCTT TAGCACGAGA AGATGTATAT TA -            #GATATAAA    180                                                                 - - CCTTATCTTA TTTAATTAAT TTAGTAAGAT TGAATTAGAG GTAAATTTTA TT -            #ACTTAATA    240                                                                 - - TAATTAGACT ACTCATAAAT ATATAAATTT AAATTTTAAG TGTTCATTCC AA -            #TATATGAA    300                                                                 - - ATCTATTGAA AATATCACGT CAACTAATAA TATAACAAAA CTATAATATA AA -            #AATAAGTA    360                                                                 - - TAAATTTTAT ATTTATAAAC AATTTTGACA TTAAATTAAA CTTAAATTTA TC -            #TCTATTAA    420                                                                 - - TAATAATATT ATAAGACAAA TTACTCTGCT AAAATACAGA AAACAATATA TT -            #TTTTTGAA    480                                                                 - - ACTTTGAAAT ATTATATTGT TGGATGATGT TGGATAATTA GAAAGGACAT AT -            #TATATATA    540                                                                 - - TGTCACGTTG AGATGAGTGG CCCATTGCAC TGAAAATGAC TGACAAATGG TA -            #CTCTCAAT    600                                                                 - - CCCATCTTAT TCTCTGTTCA ATTTTTTTCA CTTGAAAACT CTTTTTCCCT AT -            #GGAAAATA    660                                                                 - - GCAATAACTA CAATATCCTC GTTTCTTCTT GTTAGCTCTT GGCTACAACT GT -            #GTTCATCT    720                                                                 - - TCTCCACTTT CATCAATACA ATTCCAAACA GAATATACTT AGACCCTTCT GC -            #TATTTCAA    780                                                                 - - GAAAGTAGCT TGCAAATTTG CTTTGTTTCC GACATACACT TCAATATGAA AA -            #AAAAAAAA    840                                                                 - - AAAACACTTT GAGAACTTTT TAAAAAGTAT TAAGTAGGAT TTGACGGCAG AA -            #TTTTGTTT    900                                                                 - - CCATATTTAG TTGAAAATAC ATACAAAACG TATTTGAAAG TTATATTCGA TT -            #GAATTTGG    960                                                                 - - TTTTAACATA GAAAAAATTC AACCAAATTA AGTCCATACT TAAGCATTAA TA -            #TAAATATT   1020                                                                 - - TCAGTTATTC GACTTCGGTT TCACGTCTTG CCATTGTTTT ACATGTGTAA TA -            #CTTCAATT   1080                                                                 - - AATTTTTTAT GTTTTCATGT CTCTTTATCC ACTCCCTTTA TTTTTACATT AT -            #AATACCAC   1140                                                                 - - ATTCCTCCAA TACTATAATT CTTAAGATAT ATGTGAACAT TAATATCTAA TG -            #ATACATAA   1200                                                                 - - GGTAAGTTGT AAATATTCAT AGAAAAAATA AAATGACTTT TCAAGAAAAC CA -            #ACAACTAA   1260                                                                 - - ATATAAAATA TAGAAAAGTT ATTTACAATT TTGTCCGTTA ACATGTCCAG AT -            #ATTACACT   1320                                                                 - - CTCAAAAGAA AAAGTGTTAG AAAAATCATA TAAAATAGAG TTCAAATTCT TT -            #GTTAGATT   1380                                                                 - - TTTTTTACTG AACATTTAAA ATATATATTG ATATTGATTA TTCATTTTTA TA -            #AATATATT   1440                                                                 - - TTAAAATTAA CATTCAATAT ATATATTTTA AAATTAACAT TCAATATATA TA -            #TTTTAAAG   1500                                                                 - - ACACAGAAGA AACAACAAAT TCCATAAAAT TGTGAGATAA TATTTAACCC TA -            #ACTTTCTT   1560                                                                 - - ATGAACTGAG AGATTTTACA TTTATGAGAA ATGATTGTCC TGTGTTAATT AT -            #CCATGTCA   1620                                                                 - - GCTACCTAAT CACTAGAAAA GCTAATCAGA ATTCTGTGAT CTAGTCCTAC TA -            #TTCAAACA   1680                                                                 - - CTTTTAGGCC AAAGAAAATT GAAACACAAA ATACCAGTTC TCAAATACAA TG -            #AACATTAT   1740                                                                 - - TAATTATAAT TCAGTTAAAA GTCATTGATC AGAACAGCAG TGAAGGTTAG CT -            #ATAAGCGC   1800                                                                 - - GTTATAGGTG CAGGCAGAGT GTCGTGCCTA TATATACCCT TTGGAATGCA CA -            #AGTTGAAA   1860                                                                 - - CACAAAAGAA AAATG              - #                  - #                      - #  1875                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1965 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - AAGCTTCAAG TAAGTCTCTG TGATATGTAT GCAAGGGTTC GAAATGAGAA GA -             #AGGCCCTT     60                                                                 - - CAAATTCTAG GTGTACTGGA ATCTAGGAAG GATGAATTAG GAAAAGCTGA TT -            #TTGAGAGA    120                                                                 - - ATTATAAGTG GCCTTATTGA TGGTGGGTTT CGGCAAGATG CCCAACGAAT AT -            #GTGGGATC    180                                                                 - - ATGGAGGCGC AGGATTTCGA TGCATCAAAG GTTAAGGTCA ACCTTATGAA GC -            #CTGTCTCT    240                                                                 - - AGAGGACCTC GTATGAGATA GTTTAGTGGT CATGAATTGG GACATTTTAG TC -            #TTTCTCTG    300                                                                 - - CAAGTGAGTT ACAAATGTAT TACCTTATAT AGGAAGCAAT GTCTGCATGA TT -            #TATCATAC    360                                                                 - - CATGTAACAA ATAAGAATGA ATTTGTTTAT GGATTTTTCC ATTGCTCAGA TT -            #CTGAATTT    420                                                                 - - ACGCAATTTT TTTTTTCTTT TGAACTTTAG TTGTTTGTAT ATACAAATGT CT -            #TCTGTGGC    480                                                                 - - ATGTTCATGG AATTTTCATT TCCAATTATT CAATATTCTT GTGGTGTGAT CA -            #TCACTTTT    540                                                                 - - GTTAGGCAAA TCTGACAGCA CTGATGCCCC CTATCAGGAT TTTTAAACTT GT -            #ATGCGGTA    600                                                                 - - TACTATACTG ATCACAAGAT ACAAACTAAT ATAAATGGAT AGGAAATGCA GA -            #TGGGATGG    660                                                                 - - TTCAAGCTAG TCTTTAATAT TGAGATAGTA CAGAAAATGC AATGCCCAAA GT -            #AAACAACG    720                                                                 - - CTGATATTTC AAAATCACAT ATTAAAGCTA AAGTTGGTAG CAACTAGCGT GA -            #GAGCATCC    780                                                                 - - TAGTCTAGAC TGTGAATGCA GTATTTATAC ACTACAATGA TCTAAATAAG AT -            #GCTACTAA    840                                                                 - - TGCAATCATG CTTAATGTAA TATGAATTGA TCTAAAGTAG CTTGCAAATT TG -            #CTTTGTTT    900                                                                 - - CCGACATACA CTTCAATATG AAAAAAAAAA AAAACACTTT GAGAACTTTT TA -            #AAAAGTAT    960                                                                 - - TAAGTAGGAT TTGACGGCAG AATTTTGTTT CCATATTTAG TTGAAAATAC AT -            #ACAAAACG   1020                                                                 - - TATTTGAAAG TTATATCCGA TTGAATTTGG TTTTAACATA GAAAAAATTC AA -            #CCAAATTA   1080                                                                 - - AGTCCATACT TAAGCATTAA TATAAATATT TCAGTTATTC GACTTCGGTT TC -            #ACGTCTTG   1140                                                                 - - CCATTGTTTT ACATGTGTAA TACTTCAATT AATTTTTTAT GTTTTCATGT CT -            #CTTTATCC   1200                                                                 - - ACTCCCTTTA TTTTTACATT ATAATACCAC ATTCCTCCAA TACTATAATT CT -            #TAAGATAT   1260                                                                 - - ATGTGAACAT TAATATCTAA TGATACATAA GGTAAGTTGT AAATATTCAT AG -            #AAAAAATA   1320                                                                 - - AAATGACTTT TCAAGAAAAC CAACAACTAA ATATAAAATA TAGAAAAGTT AT -            #TTACAATT   1380                                                                 - - TTGTCCGTTA ACATGTCCAG ATATTACACT CTCAAAAGAA AAAGTGTTAG AA -            #AAATCATA   1440                                                                 - - TAAAATAGAG TTCAAATTCT TTGTTAGATT TTTTTTACTG AACATTTAAA AT -            #ATATATTG   1500                                                                 - - ATATTGATTA TTCATTTTTA TAAATATATT TTAAAATTAA CATTCAATAT AT -            #ATATTTTA   1560                                                                 - - AAATTAACAT TCAATATATA TATTTTAAAG ACACAGAAGA AACAACAAAT TC -            #CATAAAAT   1620                                                                 - - TGTGAGATAA TATTTAACCC TAACTTTCTT ATGAACTGAG AGATTTTACA TT -            #TATGAGAA   1680                                                                 - - ATGATTGTCC TGTGTTAATT ATCCATGTCA GCTACCTAAT CACTAGAAAA GC -            #TAATCAGA   1740                                                                 - - ATTCTGTGAT CTAGTCCTAC TATTCAAACA CTTTTAGGCC AAAGAAAATT GA -            #AACACAAA   1800                                                                 - - ATACCAGTTC TCAAATACAA TGAACATTAT TAATTATAAT TCAGTTAAAA GT -            #CATTGATC   1860                                                                 - - AGAACAGCAG TGAAGGTTAG CTATAAGCGC GTTATAGGTG CAGGCAGAGT GT -            #CGTGCCTA   1920                                                                 - - TATATACCCT TTGGAATGCA CAAGTTGAAA CACAAAAGAA AAATG   - #                    1965                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2960 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - AAGCTTGATA GATACAATTT GTATGTACCA ACTTGAGAGG AGTGTTAAAT AT -             #ATTATTTT     60                                                                 - - TATTTTATAT TTATCTTTTA TTTTTAGTTA GTTTGTTATT ATTTATTATT TG -            #TATTTTGG    120                                                                 - - GCTTAGTACA TATTTCTTCT ATTATAAATA AAAGACTCTA CGTGTATATT CA -            #ACATAAAG    180                                                                 - - GAGATTAATC TTATACATAA TTTTCACTAT ATTCAACAAC TATCATAAAA AA -            #CATGTAAA    240                                                                 - - AAGAGGCAAT TACCATTGCA TCTTTAACAA CAATTGTGAC TTTAAACTAT CG -            #TTATTACT    300                                                                 - - AGTAACAAAA TCCTATTTTT ATACATGTAA ATATTTAGGA TGAAAATTAT CT -            #CTTTCCAT    360                                                                 - - TGAATAATAA TAAACTTTGG ATAAATAAAA TTTGATCCTG TATTATTAAT TT -            #TATTTTTG    420                                                                 - - AAAAGAATGA AAATTTTAAT TTAATTTTTC ATTACATACA AATTTTCAAA TT -            #CATTAGTA    480                                                                 - - ATTATAAAAT AGTTTCATGT TTTTGTTAAA TTAGTTGTCA AAACATATTT TT -            #AATAAAAT    540                                                                 - - ATCTCGAAAA AAATGTTAAC AATAAAAAAT AGGACCTTTT GACACTCCAT AA -            #AAAAACAT    600                                                                 - - GTTTTTTTAA TCAGAAAAAC ATGTTATAAT AATCGATAAT ACTATTCTTC AT -            #ATATCAAT    660                                                                 - - GTATACATGT TAGAAATACT ATATATGTTA CTCAAACTAA TATAATATAT AC -            #TTATATTT    720                                                                 - - CAAAAATAAA AGAAGATAAA ATTATCCTAC ATATTGTTTC TTTAAATTTA CA -            #TATAAAGT    780                                                                 - - CATATTATCG TTTTGAGTAC TCACTTAAAT AATCAAACAT GGTATATCAT AC -            #AACATATA    840                                                                 - - CATATATTAG TTTACAGATA AAATTATAAC AAAATCTATC TAATTCACTT TT -            #TAAGAACA    900                                                                 - - CAAATATTTA ATTACATTTC AATATTCAAA GTAATTTGTT ATTGATATAT TT -            #AGAGGATT    960                                                                 - - CATATTAAAC ACATGTAACA AGGAAAATAT ATAGAAAATA TCGTCTTATT TC -            #AAAGTTAG   1020                                                                 - - ATAATTCATT TAACATAAGT CTTTTCTATT CTTGTCACCT AATATCTTAA TG -            #CTTATAAT   1080                                                                 - - CTATAACCCC CCCAACAATA TATCATATTT ACATAATGAT TTATACTATC AA -            #TAATATCA   1140                                                                 - - TGACTCTTGA GACATAATAT CATCTCTCAC CATACACTCC CAAAATAACA AT -            #ATCATATA   1200                                                                 - - TAACATCATA AAAGTATCCA CATGAAATAT ACATCATCAT AATACCACAC AT -            #TTTCATCA   1260                                                                 - - TAAACATACA CATATTACAT ACATGAATAC TAATCTTTCA ACACAATACC GT -            #CACATGGG   1320                                                                 - - AGAACTTAAT TTGCCTCTCG TCCCAAAGGA GAAAACCTAA AATAACAAAC AA -            #ATTTTTTT   1380                                                                 - - TTTTGTGTTA GTAAACATAC ACACTTTTTT AACACTCATA CAATTCACAT AT -            #CTAAAATA   1440                                                                 - - ATATTTAATG AAATAAATGT AAGTTAATTA AGTGCCAGTT ATCTAAAAGT GA -            #TATGCCTA   1500                                                                 - - CTAGTCAATG GATTTAGAAC ACCAAATATC CCAATTAAGT TATTAAAACA CC -            #TTAGTTTA   1560                                                                 - - AACCTTTATA TCATTAGCAC CATTATAATA AGAAAATTTG AATAACAGGA AA -            #TTAAACAA   1620                                                                 - - TTACATTTGA TCAATAATAT ATTTAAACTG CCTTGATATT TTTACCTGCT AT -            #CTCTTTGC   1680                                                                 - - ATAAAATATA TATTTGATTG TAATTTTAGA TTTTATATAT TATAAAAAAA TT -            #AGTTTTAG   1740                                                                 - - TTCTTAATTT TTTTTATTTA AATTTGACTT CTTTAATTTT TAATCATTCG TA -            #ACTTTAAT   1800                                                                 - - CTTTGAATTT CTTGAATAAT TACTAAAGTT TTAATTATAT GCAACTTTAT TC -            #AATTTTCA   1860                                                                 - - ATTTTGAAAT TATACTGAAG CACTATTTTA TTACATTTAC ATTAAAGTCC TG -            #CATTCTAT   1920                                                                 - - TCTTCTCAAT TTTCTAAAAG ACCACGCACA TTATATACTT TACCCAATCT TA -            #TTATATTA   1980                                                                 - - TGTTTAATGT AACCCAAATT ATAGATAATT GATCTTAAAA TTGAACAACA TT -            #ATGATCGT   2040                                                                 - - TAAAAACTAA AATATACAAA TTGGGTAAAA GAAAATCCAC AGACCCAAAT AA -            #TGAATATT   2100                                                                 - - ATAAAATGAG GGACTAAAAA CTACATAAAA TAATATGGAC CCAAAAAAAT AC -            #ATATTTTA   2160                                                                 - - TAAAATATAA ATTCCAGAAT TACAATTAAG TAAAAAGATA TTAAAAGATA AG -            #ATAATAAA   2220                                                                 - - TTATTTATCA AATATTTTTA ATTTAATTAT AAAATTTGTT ATTTAAATTT TA -            #TTTTTCTA   2280                                                                 - - AAATTTAAAA AAAAAACTTA TAATTAATAA GTTTAGCATA CAGGTGAGCA TG -            #TCAGTATT   2340                                                                 - - ATATAAATTA AATATGTCAA TAGTCCATTT AGTATTAGGT GTATTGTCAT AT -            #ATCAACAT   2400                                                                 - - GAAAGCAACA TGATTTAAAG AATAATAAAC TAATACATGA TTAAAACCGT TT -            #AAATTTAG   2460                                                                 - - AAATTAAGAA ACCAAGCGTA CAGAATTTAA AAGTAAATAA AAATCACATT GG -            #AAATTTTA   2520                                                                 - - AGAGGATAAA AAATACAATT AAATCTAAAT GGTTTCTAGT TAATATGTTT TC -            #ATACACAT   2580                                                                 - - AAATATCAAG AAGCAATTCA TTTTACTTGT TTATAGAATT CGGTTCTTAT CC -            #AAATTAAA   2640                                                                 - - AAGAAAATTT CTTAGGCATA CTAAATTATA TATTTGATTG AATTTAACAT TC -            #ATTTAAAA   2700                                                                 - - ATCATGTCTA TTAGGTACAA AATGATTGCT AATTAGCGAG CCCCAAGGTG TA -            #ATAAACGC   2760                                                                 - - GTAATATCAT GATGACACCT GTTACTTCTA GCTTTCGAAG ATCATAATCA TG -            #AACAGAAA   2820                                                                 - - TATACCTAAT GAACAGAAAG AAAACTCCTG TGGCAGAGAT GAACGAAGAA GC -            #AAACTTCC   2880                                                                 - - AAAGCACGGT GATGTGTCTA TATATATATT CCCATTAGCC TCAAAGACTT TC -            #ACAACACT   2940                                                                 - - TTCATCTTTC CCTTGTTAAC            - #                  - #                     296 - #0                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 3300 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - CCACCGCGGT GGCGGCCGCT CTAGACATAA TGATCTCTTT CAATGATCAC CA -             #TTAAATAT     60                                                                 - - AGACACAAAA TAGATTTGAA CTTAAGATTT ATCAAATTAA GTTTAACAAC TA -            #AAATCCAA    120                                                                 - - CCAGAGAACC ATGATCTCTA TCCACAAGTT ATTTTAGAAT GATTTGAGAA TG -            #AAATTCTA    180                                                                 - - CTAATTAAGT CATAAAAGTA TAACAAAAAA CATGAACATA TAGAAATGAT AA -            #TGAAATGC    240                                                                 - - ATTTTTTTAA CTATTCTTGC AGGATAGAAA ACATACTGCA AAGATTCCAG AG -            #AAAGTTTT    300                                                                 - - TCTCTTTACT CTTCAACCTT TTAGCTCATA TTCTTCCATG TCTAGGTATC GT -            #TCCAAGCG    360                                                                 - - AGAAGAAGTG TGTTTGTAAA AGACACTATG ACGCTCAAGT AAGGAGTGTG CC -            #TTTGATGA    420                                                                 - - TAATAAATAT TTTAATAATG AACACATAAT TAATTACCTC GTGAACAAGA CT -            #ATTTATAT    480                                                                 - - TAGGTTTATG GGTCCTTACC TGTTGGGCTT GGATTACATA GATAATCATC AT -            #GGTTAATT    540                                                                 - - TGTTTAGTGA TCTTGCTAAT ACTTTTAACT CTTAACCTTT ACTGATCCTT AC -            #TATTACAA    600                                                                 - - TGTGATCTTA AACATTACAA AATGAAATAA TGTTAGGTAG GTGTTCATGA AT -            #ATTTAAAA    660                                                                 - - TGATTCTTGA TCGGTATGAG CCAAAATCAT CTCTGGTACA TATAAATAGA GA -            #TGAGTTTA    720                                                                 - - GTCATTACAT ACCCACATAA TGTTAAGTAG ATGTTTACAT ATGATTGATA AG -            #ATAACCTC    780                                                                 - - TCGTATATAG GTTGAAATGG TCTTTGATAC ATGTAATAAC ATTAGATGTT AA -            #TAGTTAAA    840                                                                 - - AATTGATTAA AATAAAATTA CATATAATAA TTTATTTTGA TACATATTGC CA -            #GACCTCAT    900                                                                 - - TTAAAACGCA CCCAAAAACC TTCTGAACGG ACGTCAGGTG TCAAGCGAAG AG -            #GATCCGGA    960                                                                 - - AATCAGATAG TGGAAGGCAG GTGTCGGCAG ATGAGCGGAC GCTCGTTTTG AC -            #GTGGGAAG   1020                                                                 - - CAAAACTTGA TTTTTCAGAA AATTCACGTC ACACTCTCTG CATGCACCTT CT -            #TCCCCAAA   1080                                                                 - - CTCTGAAAAT TTTATTTCTC CTCCTTCTCA CTAAAAACTC TCCCTTCTCT CT -            #ATAAAATA   1140                                                                 - - TCATCATTTG TTGATAATTT TGATGTTCGT TTTGAAGTTT TTTTATTATT AT -            #TTAATTAT   1200                                                                 - - AGTAATATCT CCTTCTTAAA TTCCTTAAAT AATATCTATT TATTCATGTT TT -            #CGTTATTG   1260                                                                 - - TCGATATATT CTAACTACAA AACTATCTTA AATACTTAAT AATGTAAAGT TA -            #AGGTAAGA   1320                                                                 - - TAGCGAAAGC AAAGGTAAAT GTAAATCTAA AAATAAAACA AACTTTGTAT TT -            #AGACATTA   1380                                                                 - - ATAATATATA TAAAAAATAC CCTTATATAT AATGGATTCT ACGTTTTAAG GT -            #TAAGGGTA   1440                                                                 - - TTTTAATAAT TTTCATTCTC AAAACTAAAA AAAAAAAAAA AAAAAACCTC AT -            #TTTCAAAA   1500                                                                 - - CTAAAAAAAA AAAAAAACCT CCAAACCCTT AGTTACCTCT CTCATTCCTC TC -            #AACCCTTT   1560                                                                 - - CTCTCTCATC TCTCCCACTC CAACCTTTTC TCTGTCATCC CTACTGTAGT CC -            #CAATTGAA   1620                                                                 - - AAAATCAGAA ACTCTAGCCC CAATTGAAAA AATCAGAAAC ACTTGCCGTT AA -            #ATTGCCTT   1680                                                                 - - TGTAAAGAGT TGAGTCATTG ACATATTCAC CTTCAGGAAA AGGTTCACTC AA -            #GATCTCTT   1740                                                                 - - CAATTTCACC ATCTTCATTA ACCTCTCTAA TTTCATCATC TACATGTGTT GA -            #ATCATCAT   1800                                                                 - - CTCTAAAAAA TTATAAAATG AAAAGTCATT ATAAAATCAT TTTTTGTAAG AA -            #ATTGTTTA   1860                                                                 - - ACGAGTGTCT CTGATTTTTT CCACGCCAAT TACCAATTCC TTTGATGTTA TT -            #ATGCTTGT   1920                                                                 - - GAAAATTAGA TAAAATTAGA TAAAATTAGA TAAGACAAAA ATTATAAAAT GA -            #AAACTCAT   1980                                                                 - - TATAAAATCA TTTTTTGTAA GAAATTGTTT AACAGCGAGT ATTTCTGATT TT -            #TTCCAGGT   2040                                                                 - - CAATTACCAA TTCCTTTATA CTTGTGAAAA TTGGATAAAA TTAGATAAGA CA -            #AAAATTAT   2100                                                                 - - AAAATGAAAA CTCATTATGA AATCATTTTT GTAAGATTGT TTAACGACAC AT -            #GTTTCTGA   2160                                                                 - - TTTTTTGAAT TAGGGCTATA GTAGGGATGA TAGAGAAAAA GTTGGAGTGA GA -            #GAGATGAA   2220                                                                 - - AGAGTGAGGA TTGAGAGAAA TGAGAGAGGT GAATAAGAGT TTGGGTGTTT TT -            #TTTTAGTT   2280                                                                 - - TTGAGAATGG AAATTATTAA AATACCCTTA ACCTTAAATT TAGAATCTAT GA -            #TATATAAG   2340                                                                 - - GGTATTTTTG TCTACTAAAA TCTGATACAT ATTACTCAAA TGTACCAACT AA -            #AAAGAGAC   2400                                                                 - - GTACACGCGT TACCCAACCC CATATATATA TATATATTAG CCTCCCAAAC TA -            #TCTTAAAT   2460                                                                 - - AAGGTAAAGT TAAGGTAAGA CAGCGAAAGC CATAAGTAAA TGTAAATCTA AA -            #AGTAAAAC   2520                                                                 - - CAATTTAGTT TTTAGACATT ACGAGTATTC AGGCATTCAT AATTATGGTA CA -            #ACTTTTTA   2580                                                                 - - ATAAAGAAAT AAAAAGAACA ATTCATTATA TACACAAAAA AAGTTACATA CA -            #CTGAACTT   2640                                                                 - - ATCACTTATT TCGTACACAC AAAAATTATT TATATTTTTA CATAAATCCT AT -            #CTAGTCAG   2700                                                                 - - TTTTCTCCAT TAAAATATTA TATAAAAATA TATAAATATA ATAATAAAAT TT -            #AAAATACA   2760                                                                 - - CCTCTTTGAT TTGCAACGAG CCACCAGAAG GAGAGATTGT TAATTTAAAC GG -            #AGTAAATA   2820                                                                 - - ATCATCAAGT GCCACGAAAT AGTTACATAA TCACGAAGTT ATCTACAAAA AA -            #TAGCCTAA   2880                                                                 - - AATGCATTCG AAAATTTATC ATTATTGCAA ACAACAATAC TCTAATCTGA AA -            #GAGATTGA   2940                                                                 - - TGATTACAAA GATTAGCTAG CAGTCAATTT AAATAAACGC GTAATAGTCT CT -            #CTATTAGT   3000                                                                 - - TGTTTCCAAC ACAAAATCCT AACTAAAGCA AATGCATGAT TCTTTGTCTT CA -            #TCTCTCTC   3060                                                                 - - TCATCTGACA TAAAACAAAT CTTAAATATA TATCATTAAT CATTATAACA AG -            #CATAAACT   3120                                                                 - - TGATCGTTTT TGTTAAATGA TGAAGCATGT ATTATTGAAT TAAATATAAA TT -            #TATGTTGA   3180                                                                 - - ATATTTAAAA AGATAGAAAG TAGAGGGAAA GAGAGAGGAA GAAGGGTATT GG -            #GCTAGGTG   3240                                                                 - - CAGTGCTTAT ATATACCCTT TTCTTAGCCA TTAGCTTCCA CAAACAGATA AA -            #CACAGAAA   3300                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1127 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - TCTAGATGGT TTCACCCAAC TACATGTTTT GTTCTGTTTT GCTTTGGTTT CA -            #AACTTGTG     60                                                                 - - ATAAAAGCAA CGCGTTGAGT CTGTTTGTCA ATTTTGTTCG ATTTCAGATT CT -            #CTGTGGAT    120                                                                 - - GGAACTCCAA TAAGGGAGTT CAAGAACATG GAGTCAAAGG GTGTTCCATT CC -            #CCAAAAAC    180                                                                 - - CAAGGCAATG AGGATATACT CAAGCCTTTG GAATGCAGAT GATTGGGCCA CA -            #AGGGGAGG    240                                                                 - - GCTTGTTAAA ACCGATTGGA GCCAAGCTCC ATTCACGGCT TCATACAGAA AC -            #TTCAATGC    300                                                                 - - CAATGCTTGC ACTGTGTCCT CTGGAACTTC TTCTTGTTCA AACTCTGTCT CT -            #TCTCCCAA    360                                                                 - - TGCTTGGCTC TCGGAAGAAT TGGACTCTAC TAACCAGGAG AGACTGAAGT GG -            #GTACAGAA    420                                                                 - - GAATTACAAT GATCTACAAC TATTGCACCG ACGCCAAAAG ATTTCCACAG GG -            #CCTTCCTA    480                                                                 - - CAGAGTGCAA CACTGCCTAA TTTTTCTTAT CAATCCTTTC CATGCTCCAC TT -            #TCTTTTTT    540                                                                 - - ATTTCTTCTG TTGTACTTTC CATCATGATC AATTCTTTTA TTCATTGTAA AA -            #CATTGCTA    600                                                                 - - TCATGATAAG TTTTCTTAAA TATTTGCATA AGAAACTTGC CGTATAAATC GT -            #CTATAAGC    660                                                                 - - AGGAAACTAA AATAGTCCAG GAAATCGAGA ATCGAGAAAC GAGAATTTCC AG -            #GTCACCAA    720                                                                 - - CCTGTGAAAA TTGTTTTTGA TCTTCGATAA AAGTATTAGT TAATTAAAAA AA -            #CACAAGAT    780                                                                 - - TGTTGAAAAT ATTAAATAAT AGAAACCATG TACTGTGTAT GGCGGTGTCT CC -            #TTATATAA    840                                                                 - - ATTTCATGCA GAAACGCGTG AAATGATTGG TGTGGGCGTC CATTTACAAC AA -            #CAAAACTT    900                                                                 - - ACTACTTTTT CATTCTTCAC CAGCTGTCTA CAACTAATTC AAAAGTTCAC AA -            #CCTACCTT    960                                                                 - - TTTCTCACTT TCCTCTTATC TACCAATCTC TCTTTTTTTC TCCTATAAAT AC -            #CATCCTTT   1020                                                                 - - GCAGTATCAA CCAACATTCT CACAAATAAC CAAAAACAAT TTCACTCAGT TT -            #CACACAAA   1080                                                                 - - ACAATTCTGC ATGGCATTTT CAAGACTTTT ACTGTTAACA TGCATTG   - #                  1127                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1406 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - AAGCTTGTTA AACTGATTTA AAAGTTCGTT TTTTAATATA TCAGAGTGTT TG -             #ATAATTAT     60                                                                 - - GAAAGTAACT TATTTTAAAT TAAATATGAT TTATTTTTAG CCAAAAGCTA AA -            #AGTAAGGT    120                                                                 - - AAAGAGTGTT TTTTTTTCTA ACTTGAAAGT TATTTTATGT TGACCAAATA TA -            #CAAGTATC    180                                                                 - - TTTTTGCCTT AATTCTTTTA TTTTTTGTTT TTTATTTTTT ATTATTATAA GT -            #TGCGCATA    240                                                                 - - TAAAATTAAC TTAAGTAATT AATTTATATA TTTGTCTTAT GAATAATTTG TG -            #ATGATAAA    300                                                                 - - GAAATATATG AATGATCAAA AATACTATTA CTTATTGATT AAAATATAAA TT -            #AATTTGTT    360                                                                 - - CTAACTCTTT TAAGTATAAA AAACTTAAAA TTAAACAAAT TTTTTTCATG TT -            #AACCAATT    420                                                                 - - TAAAGGTATT TCAAATATTT TTATTTTAAA AAGAAGGTGT TTCCCCGCAT TT -            #ATTTGCAA    480                                                                 - - AACACATCAA GAATCTTTTT TCAACTTCGA CACTTTTATT CAAACATATG AA -            #TAATTATT    540                                                                 - - TCAAATATAA TTTTTAGCAC TTTAAAAATC TTTTTTTAAT TCAATCTAAA TA -            #GGCTCTTA    600                                                                 - - ATAATTTTTT AAATTAATTA GACTTATTTT TAAATTTAAT AATTATTTAT AA -            #AAAAATCG    660                                                                 - - TATAAAATCG AAAAAAACAA AAGCACGCGC TATTAGGTCG AGTGAGATGG AT -            #GGGGTCAT    720                                                                 - - AAAATTTTGC TCCTCGGTCT GAGGGTGACA AGCCTTTTCT CTGATACGGG CA -            #TGTGCATG    780                                                                 - - TCCCCGTTAA TTACTCCCCC AATGTGCAAT TACCCACTAA CTCTAACCCC TC -            #TTTTGGAC    840                                                                 - - AATTATTTGA AAGGCTTTAA TTTAATTATT TTTTTGTTTT TCATTCCATC TA -            #TACTTATA    900                                                                 - - TTAAAGTTGA ATCAAATTTA GAATTACACT TGTATTTAGC ACTAAAGTGC TA -            #TATAATAA    960                                                                 - - AAAATGACTA TGTACTCAAG AAAAATTAAA TTTGAAATCA ACAGAAGAGT CA -            #TAATTTTT   1020                                                                 - - AATAAAGAAA TTTAAAATTT ATAAAAATAA ACACAAAAAA TGTCTCAAAG GA -            #GATTAGAT   1080                                                                 - - ATCTATTAGA ATATTATTGT AATAAAATAT AAATAATATA ATTTTGCATA TT -            #CGAAGTTT   1140                                                                 - - CTGATTAAGG ACGAAAGAAT AATCGTGGCT GCACAATAAC CTTTGTTGGT GA -            #AAGGACAA   1200                                                                 - - ATTTCAACCA CCCAAAATCT GAAAAATCTA ACTTTGTTTC AACTTTCAAC CA -            #CAAGTCCA   1260                                                                 - - ACTCAGTCCC TTTTACACCT ATAAATAACC AGTCACTACA CTTCCATTTT CC -            #TCACCCCC   1320                                                                 - - ATTGGGCCAT ATTCATCATT CTCTAAAAAA AGAAAAAAAG AAAAATACAC AA -            #ACACTGGT   1380                                                                 - - CTCTGATTGG ATTTGTTTTT CTCACC          - #                  - #                1406                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 800 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - TCTAGAGTTA GATCCCGAAT AATTATCTTA CACCTATCCT ATCAAAACTC TA -             #TTTTCTCT     60                                                                 - - CATTGATAAC CTTCTTTGCT TATTCCTTGT TTCGATAATC ACTAGTCAAT AG -            #ATTTAGAT    120                                                                 - - TCGTAGTTAA TTTTAGTATT AATCATATAA ATCTCAACTG TTGATCCTCT TG -            #GATAGCAA    180                                                                 - - TCAAGGTAGA AACTACGAGA ATACTGTTTA AATCCAATCC TTGTGGATAC GA -            #TATTATAC    240                                                                 - - TATATTATCT TTGATTATTG AGCATAATTA AGTGTGTGTT TTGCGTTCGT TA -            #CAAAAGTC    300                                                                 - - AAGTTTTCTT GAAAATAAAA ATTTCAAATT ATGTTATACT ATTTTATAAT AG -            #TACTTTAC    360                                                                 - - TATAGCAGTC AAAAAATATT TGGAACAAAA TGAAATTGTT ATAGAGGGGT TT -            #AGACATTT    420                                                                 - - TAAGCGATAA TTAAAAGTGA AAAGCACGCG CTATTAGGTC GAGTGAAATG AA -            #TGGGGTCA    480                                                                 - - TATAACTTTC CTCCTCGGTC TGAGAGTGAC AAAGCTTTTC TCTGACGCGG GC -            #ATGTGCAT    540                                                                 - - GTCTCCGTTA ATTGCTCCCT CAACGTGTAT TACCCAATAG ACACCTCCCA AT -            #TATTTAAA    600                                                                 - - AGGCCAAACA CAACCACCGA AAATCTCACT TTGTTTCAAC CCTGTGTTGA CG -            #ACCACAAG    660                                                                 - - TGATTCCTGT TCCTGCCCCT TTACACCTAT AAATAATCAG CCATTTCCCT TC -            #CATTTTCC    720                                                                 - - TCACCCCCAT TGGGCCATAA TCCATTCCCA AACAAAGATA CATAGTTGTT TC -            #TGATTGGC    780                                                                 - - TTAGCTTTAG AACTTTCACC            - #                  - #                      - #800                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1138 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - GCTTGACTTT AATCAAACCG GTATATATAA ACTAAAAAAA CGAAGGGAGT AC -             #TACATACT     60                                                                 - - AGCTTTATAA TACTAGTACT GGATAAAATC TGCCACAGAA AGATTTTAGC GG -            #GGAGAAGG    120                                                                 - - AGCATCATAG ACTGACTGAA TGATAGAAGT GTTTTCACCG GCGGTGCATT GC -            #TTTCAATC    180                                                                 - - AATCCATTGA AATGGAGTCC AGCTGCTTAC CCTAATCTAA TCACAGGATG AG -            #CCCATGGA    240                                                                 - - TCTAGCTGCA GTACCTCGAC TCCACCGGAA AGGAGCGGGC CCGTGTCGGT AG -            #CGTTGCTC    300                                                                 - - CGGCTGGGTC CAGCACGACC CGACCGCGGC ACGCGTGGCG TTGGATTTGG AG -            #ATTCGGGC    360                                                                 - - TCCTGATTGT GATGCGAGTC TGCAACATGC ACAGCCATGT GACCTGCATT GA -            #TTCCTGCC    420                                                                 - - AGCCACTGTG CTGTGTGTGA GACCTGACCT GCACAAGAAC GGATCAAAGC TG -            #GGGCCGGC    480                                                                 - - CCTTCGCGGC ATCATCAACC TCTCAAAAAC TCGTGTAAAA ACAGGTTCAC AA -            #AATAACTC    540                                                                 - - ATCTGAAACA ACTCCTCAAA ATCTGACGCA GAAATGAGCC TTCTATAGAG TA -            #GAAGAAAC    600                                                                 - - AGCAAATGCT GCAAAAGGCG AAAAGGCTGG TCCGTCGAAT GAAATTCTGA TA -            #CTATTGCC    660                                                                 - - TCGATTCAAC ATATATATAC TTATAATCCA AACAAGAAAT CGTACTGTAC TC -            #CGATCCGA    720                                                                 - - TGGCAAATAA ATCAGTGGCA ATGGCAGCAA GTTGCGAGGT GTGCATGATC CG -            #TGGATCAA    780                                                                 - - TCAACAATGC TTGATTTGCT CGCACTGGGC CAACCTGACA CGCACAAGAC AA -            #GCATTGCA    840                                                                 - - CCTCGCAAGC ACCTCACTCC ACAGCGTCCC CATGCACTGG ATGCAGCTGG CT -            #CACTCATC    900                                                                 - - ACTCGATTGC CATCGCTCGA TCCATCATGT TCATTTAGTG CCACGTCAAA AC -            #AGATTATT    960                                                                 - - TTTATTTCGC CAAGCAACCA ATAATGTACT CCAAGAACCT ACGTACAGTG AG -            #CTCACACT   1020                                                                 - - AGCTATAAAT ACACACAGGC TTCTTCGTCT TCGCATCCAC CACTCGCCCA TT -            #GTTTGTAG   1080                                                                 - - TACCAACCAG CCAAGCCAAG AAGTAACAGA GAAGGAGGAA GAGAGGCCGG CC -            #GGCGAA     1138                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 173 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - CTTCAGCATG CACATCAAGC TCGTCGCCGG CGACTCCGCC GGCACCGTCA CC -            #GCCTTCTA     60                                                                 - - CCTGTCGTCG CAGAACTCGG AGCACGACGA GATCGACTTC GAGTTCCTGG GG -            #AACAGGAC    120                                                                 - - GGGGGAGCCG TACATCCTGC AGACGAACGT CTTCTCCGGC GGGAAGGGGG AC - #C               173                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 98 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - TGAAGCTCGT CGGCGGCGAC TCCGCGGGCA CCGTCACGGC CTTCTACCTG TC -             #GTCGCAGA     60                                                                 - - ACTCGGAGCA CGACGAGATC GACTTCGAGT TCCTGGCA      - #                      - #     98                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1130 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - GGTTTCCCTT GTTAACATGG CTTCTCCTTT GTTGATTTTG TGTCTTGTTC TG -             #GTTTCGCT     60                                                                 - - AGCCTCTGCT GCACTCTGTG CGGCCCCACG GAGACCAGTG GATGTTCCAT TT -            #GGCAGAAA    120                                                                 - - CTACATTCCC ACATGGGCTT TCGATCACAT CAAATACTTC AATGGGGGTT CT -            #GAGATTCA    180                                                                 - - ACTTCATCTT GACAAGTACA CTGGCACTGG TTTCCAAACA AAAGGGTCCT AT -            #CTGTTTGG    240                                                                 - - TCACTTCAGC ATGAACATAA AGATGGTTCC TGGTGATTCA GCTGGCACAG TC -            #ACTGCTTT    300                                                                 - - TTATTTATCA TCTCAAAACG CGGAGCACGA TGAGATAGAC TTTGATTTCT TG -            #GGGAACAG    360                                                                 - - AACAGGACAA CCTTACATTT TACAGACAAA TGTGTTCACT GGAGGGAAGG GT -            #GACAGAGA    420                                                                 - - GCAAAGAATC TATCTTTGGT TTGATCCCAC AAAAGCGTAT CACAGATATT CT -            #GTACTATG    480                                                                 - - GAACATGTAT CAAATTGTAT TCCTAGTGGA TAACATCCCA ATCAGGGTGT TC -            #AAGAACCT    540                                                                 - - GAAGGAGTTG GGAGTGAAGT TTCCCTTTAA CCAACCGATG AAGGTTTACA AC -            #AGTTTATG    600                                                                 - - GAATGCTGAT GATTGGGCCA CAAGGGGTGG TTTGGAGAAA ACAGATTGGT CA -            #AAAGCTCC    660                                                                 - - ATTCGTAGCA GAGTACAAGG GGTTTCATGT TGATGGGTGT GAGGCTTCAG TG -            #AATTCAAG    720                                                                 - - GTTCTGTGCC ACACAGGGTA AGAGATGGTG GGATCAAACA GAGTTTCGTG AT -            #CTTGATTC    780                                                                 - - CTTTCAGTGG CGAAGACTCA AATGGGTGCG TCAGAAATTC ACCATCTACA AC -            #TACTGCAC    840                                                                 - - TGACAGAACC CGCTACCCTC AACTTCCACC AGAATGCAGA AGAAACCGTG AC -            #ATTTAAAT    900                                                                 - - TTTCATCTGC TGTTTTTATC ACTTATTTCT GTGTTCTACA ACAACTTTCT CA -            #CTGCATTC    960                                                                 - - ATCATTTACC AGTTACCATA CTTTATTCCT ACCATTATTT ATTACCATTG TA -            #TTGTTTGG   1020                                                                 - - AATGTGTAAT TAAGGCCTTG GGGTCTGAAT ACAGAGGAAA CTCTATAATA AA -            #ACTACGTA   1080                                                                 - - TGTTATGTAA TTCTATTCTT ATACTTGGGC ACCACCAATA ATGTAATATT  - #                1130                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1068 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - CAGAAAATGG TTTCTTCTTT GTGGACCGTG TCTCTGATAT TGGCATCGCT GG -             #CCTCTGCA     60                                                                 - - GCAGTTTGTG CCAACCCGAG GAGGCCAGTG GATGTACAAT TCGGTAGAAA CT -            #ACGTTCCT    120                                                                 - - ACATGGGCTT TTGATCACAT CAAATACTTC AATGGTGGTT CTGAGATTCA AC -            #TTCATCTT    180                                                                 - - GACAAGTACA CTGGTACTGG CTTTCAGTCC AAAGGGTCAT ACTTGTTTGG CC -            #ATTTCAGC    240                                                                 - - ATGTACATAA AGATGGTTCC TGGAGATTCA GCTGGCACAG TCACTGCCTT CT -            #ATTTATCT    300                                                                 - - TCTCAAAACG CGGAGCACGA TGAGATAGAC TTTGAGTTCT TGGGGAACAG AA -            #CAGGACAA    360                                                                 - - CCTTACATTT TGCAAACAAA TGTGTTCACC GGAGGAAAGG GTGACAGAGA GC -            #AAAGAATC    420                                                                 - - TATCTCTGGT TTGACCCCAC CAAAGCATAT CACAGATACT CTATTCTCTG GA -            #ACTTGTAT    480                                                                 - - CAGATTGTGT TCTTTGTTGA CGATGTGCCG ATCAGAGTGT TCAAGAACAG CA -            #AGGACTTG    540                                                                 - - AGAGTGAAGT TTCCATTCGA CCAACCTATG AAGCTATACA ACAGTTTGTG GA -            #ATGCTGAT    600                                                                 - - GACTGGGCAA CAAGGGGTGG TTTGGAGAAA ACAGATTGGT CGAAAGCTCC TT -            #TCGTAGCA    660                                                                 - - GGGTACAAGG GGTTCCACAT CGATGGGTGC GAGGCCTCTG TGACCGCTAA GT -            #TCTGCGAC    720                                                                 - - ACACAGGGCA AGAGATGGTG GGACCAACCA GAGTTTCGTG ACCTTGACGC CG -            #CTCAATGG    780                                                                 - - CAAAGACTCA AATGGGTGCG TCAGAAATTC ACCATCTACA ACTACTGCAC TG -            #ACAGAAAA    840                                                                 - - CGCTACCCTC AACTTTCCCC TGAATGCAGT AGAGACCGCG ACATTTAAAT TT -            #TCACATAC    900                                                                 - - TTCTGTTACC ATTTACTTTT ACCAGATTGT TGTCACTTTC ATGTACAATT TT -            #ATATCACG    960                                                                 - - TCAAATCTAT CCATTGCCAC TTTATTTATG AATTGAAATT TGCTTCAGAT AA -            #AAAAATTA   1020                                                                 - - TAAATAAACA CAGTTTTTCT TAGAAAAAAA AAAAAAAAAA AAAAAAAA  - #                  1068                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - - GATGAGATAG ACTTGAGTTC TTGGG          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1017 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - - GAAACAATTT CACTCAGTTT CACACAAAAC AATTCTGCAT GGCATTTTCA AG -             #ACTTTTAC     60                                                                 - - TGTTAACATG CATTGTTGGG TATTTTCTGA TTGCCTCTGC ATCCAATTTC TA -            #TCAAGATT    120                                                                 - - TTGAAATAAC CTGGGGAGAT GGTCGTGCCA AGACACTAAA CAATGGCGAC CT -            #TCTTACTT    180                                                                 - - TGTCTCTTGA CAAAGCCTCT GGCTCCGGCT TTCAGTCAAA GAATGAATAC CT -            #TTTTGGCA    240                                                                 - - AAATTGACAT GCAACTCAAA CTAGTCCCCG GCAACTCTGC TGGCACCGTC AC -            #TGCCTACT    300                                                                 - - ATCTGTCTTC AAAAGGAGCA ACGTGGGATG AGATTGACTT TGAATTCTTG GG -            #GAATTTGA    360                                                                 - - GCGGTGATCC TTACATTCTC CACACCAACG TGTTTAGCCA AGGCAAGGGT AA -            #TAGGGAGC    420                                                                 - - AACAATTCTA CCTCTGGTTT GACCCAACTG CTGATTTTCA CACCTATTCC AT -            #CCTCTGGA    480                                                                 - - ACCCTCAACG TATTGTATTC TCTGTGGATG GAACTCCAAT AAGGGAGTTC AA -            #GAACATGG    540                                                                 - - AGTCAAAGGG TGTTCCATTC CCCAAAAACC AAGCAATGAG GATATACTCA AG -            #CCTTTGGA    600                                                                 - - ATGCAGATGA TTGGGCCACA AGGGGAGGGC TTGTTAAAAC CGATTGGAGC CA -            #AGCTCCAT    660                                                                 - - TCACGGCTTC ATACAGAAAC TTCAATGCCA ATGCTTGCAC TGTGTCCTCT GG -            #AACTTCTT    720                                                                 - - CTTGTTCAAA CTCTGTCTCT TCTCCCAATG CTTGGCTCTC GGAAGAATTG GA -            #CTCTACTA    780                                                                 - - ACCAGGAGAG ACTGAAGTGG GTACAGAAGA ATTACATGAT CTACAACTAT TG -            #CACCGACG    840                                                                 - - CCAAAAGATT TCCACAGGGC CTTCCTACAG AGTGCAACAC TGCCTAATTT TT -            #CTTATCAA    900                                                                 - - TCCTTTCCAT GCTCCACTTT CTTTTTTATT TCTTCTGTTG TACTTTCCAT CA -            #TGATCAAT    960                                                                 - - TCTTTTATTC ATTGTAAAAC ATTGCTATCA TGATAAGTTT TCTTAAATAT TT - #CATAA          1017                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 588 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                              - - GATGAGATTG ATTTTGAGTT CTTGGGGAAC CGTAGTGGTC AGCCTTACAC AG -             #TTCAGACA     60                                                                 - - AATATCTACG CTCATGGAAA AGGGGATAGA GAGCAAAGGG TGAACCTCTG GT -            #TTGATCCT    120                                                                 - - TCCGCGGATT TTCACACCTA CACTATCATG TGGAATCATC ACCATATTGT GT -            #TCTACGTT    180                                                                 - - GATGATTTTC CCATTAGAGT GTACAAGAAC AATGAAGCGA AGGGAATCGC AT -            #ACCCAAAG    240                                                                 - - ATGCAGGCTA TGGGAGTGTA TTCGACGTTG TGGGAAGCTG ATAACTGGGC AA -            #CAAGAGGG    300                                                                 - - GGATTGGAGA AAATCGATTG GAGTAAGGCA CCATTTTATG CATATTACAA GG -            #ACTTTGAC    360                                                                 - - ATTGAAGGGT GCCCAAGTCC AGGACCTGCT AACTGTGCCT CTAATCAAAG TA -            #ATTGGTGG    420                                                                 - - GAAGGAGCTA CATACCAAGC TCTTAATGCC ATGGAAGCTC GAAGGTACAG GT -            #GGGCTCGT    480                                                                 - - CTTAACCATA TGATCTATGA TTACTGCCAA GATAAGCCAA GGTACACGGT CA -            #TCCCACCA    540                                                                 - - GAGTGCCTTG CCGGCATTTA AACCCAAGAA CTCAAAATCA ATCTCATC  - #                   588                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 854 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                              - - CTGTCTTTGG ACAAAGTTTC TGGCTCTGGT TTTCAATCTA AGAAAGAGTA TC -             #TCTTTGGG     60                                                                 - - AGAATTGATA TGCAAATCAA ACTTGTTGCT GGAAATTCTG CTGGAACTGT CA -            #CTACATAC    120                                                                 - - TATTTATCTT CTCAGGGACC CACACATGAT GAAATTGACT TTGAATTCTT GG -            #GAAATGTT    180                                                                 - - ACTGGTGAAC CTTATATTCT CCACACAAAC ATTTATGCCC AAGGCAAAGG AA -            #ACAAAGAG    240                                                                 - - CAGCAATTTT ACCTTTGGTT TGATCCTACC AAGAACTTCC ACACCTACTC AA -            #TCATATGG    300                                                                 - - AAACCCCAAC ATATCATTTT TTTGGTCGAC AACACACCAA TAAGAGTTTA CA -            #AGAATGCT    360                                                                 - - GAATCCATTG GTGTGCCATT TCCCAAGAAC CAGCCCATGA GAATTTACTC TA -            #GCCTTTGG    420                                                                 - - AATGCTGATG ATTGGGCAAC AAGAGGAGGC CTAGTGAAAA CTGATTGGTC TA -            #AAGCACCA    480                                                                 - - TTTACAGCCT ACTATAGAAA TTTCAATTCT CAAACTTTTA GCAGTTCACA AT -            #TTTCAAAT    540                                                                 - - GAAAAATGGC AAAATCAAGA ACTTGATGCC AATGGCAGAA GAAGACTCAG AT -            #GGGTGCAG    600                                                                 - - AGGAATTTCA TGATTTATAA TTATTGTACT GATTTTAAGA GGTTTCCTCA GG -            #GTTTTCCT    660                                                                 - - CCAGAATGCA AAAGATTTTG AGTGATATTA GTTGGTTTTT GTGTAATTCT TT -            #GATGTGTT    720                                                                 - - TGTGGTTTTA TTTTGTTAGA TTATAGCAAC CAAAATAAAT GTATTTTTCT CG -            #TTTTATTT    780                                                                 - - TGTATCTTTT TCGAAGCTTG TAGTTCATCT TGTATCTAAT TTGTTTGATA TC -            #CTTTATGA    840                                                                 - - TAAAAAAAAA AAAA              - #                  - #                      - #    854                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                              - - ACACAAAATA CCAGTTCTCA            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 227 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                              - - GAATTCTGTG ATCTAGTCCT ACTATTCAAA CACTTTTAGG CCAAAGAAAA TT -             #GAAACACA     60                                                                 - - AAATACCAGT TCTCAAATAC AATGAACATT ATTAATTATA ATTCAGTTAA AA -            #GTCATTGA    120                                                                 - - TCAGAACAGC AGTGAAGGTT AGCTATAAGC GCGTTATAGG TGCAGGCAGA GT -            #GTCGTGCC    180                                                                 - - TATATATACC CTTTGGAATG CACAAGTTGA AACACAAAAG AAAAATG   - #                   227                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 290 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                              - - ACTCTTTGTA ATTTTATCGA AGAGTTAGTG TGCAATAGAA ATTTAACATT GA -             #GTATTTAC     60                                                                 - - AATTGTTAAA ACTATACATT CACTTCATTT TCATGCATTT ATAAACATTT CA -            #ATTTCAAT    120                                                                 - - TTCATGTTAA AATCAACTCA AAGTAATACT CAAATCTTAT TCCTAGTGAC TT -            #TAATATAT    180                                                                 - - TGTTAACTTA TCAAGTTTCA ATTCCTTCAA TCATCAACAA GCAATCAAGA AT -            #TAAGTTCA    240                                                                 - - AGAGTCTTAA GATTACTAAT AAATCATGTT CTATCCCTAG ATATAAGCTT  - #                 290                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1654 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                              - - AAGCTTTTTG CACATATTTG CAGCAGTAGA CAATGCCACT CGCTGAAAAA TA -             #TGATCTCC     60                                                                 - - CAGAATTTTG GCACAAAAAA TATATCCTAA CTAATATTTG ACTCTATCTA AG -            #ATACCACC    120                                                                 - - TGACATCAAA TGTTTCAATT TTATAGTCTT TAGCACGAGA AGATGTATAT TA -            #GATATAAA    180                                                                 - - CCTTATCTTA TTTAATTAAT TTAGTAAGAT TGAATTAGAG GTAAATTTTA TT -            #ACTTAATA    240                                                                 - - TAATTAGACT ACTCATAAAT ATATAAATTT AAATTTTAAG TGTTCATTCC AA -            #TATATGAA    300                                                                 - - ATCTATTGAA AATATCACGT CAACTAATAA TATAACAAAA CTATAATATA AA -            #AATAAGTA    360                                                                 - - TAAATTTTAT ATTTATAAAC AATTTTGACA TTAAATTAAA CTTAAATTTA TC -            #TCTATTAA    420                                                                 - - TAATAATATT ATAAGACAAA TTACTCTGCT AAAATACAGA AAACAATATA TT -            #TTTTTGAA    480                                                                 - - ACTTTGAAAT ATTATATTGT TGGATGATGT TGGATAATTA GAAAGGACAT AT -            #TATATATA    540                                                                 - - TGTCACGTTG AGATGAGTGG CCCATTGCAC TGAAAATGAC TGACAAATGG TA -            #CTCTCAAT    600                                                                 - - CCCATCTTAT TCTCTGTTCA ATTTTTTTCA CTTGAAAACT CTTTTTCCCT AT -            #GGAAAATA    660                                                                 - - GCAATAACTA CAATATCCTC GTTTCTTCTT GTTAGCTCTT GGCTACAACT GT -            #GTTCATCT    720                                                                 - - TCTCCACTTT CATCAATACA ATTCCAAACA GAATATACTT AGACCCTTCT GC -            #TATTTCAA    780                                                                 - - GAAAGTAGCT TGCAAATTTG CTTTGTTTCC GACATACACT TCAATATGAA AA -            #AAAAAAAA    840                                                                 - - AAAACACTTT GAGAACTTTT TAAAAAGTAT TAAGTAGGAT TTGACGGCAG AA -            #TTTTGTTT    900                                                                 - - CCATATTTAG TTGAAAATAC ATACAAAACG TATTTGAAAG TTATATTCGA TT -            #GAATTTGG    960                                                                 - - TTTTAACATA GAAAAAATTC AACCAAATTA AGTCCATACT TAAGCATTAA TA -            #TAAATATT   1020                                                                 - - TCAGTTATTC GACTTCGGTT TCACGTCTTG CCATTGTTTT ACATGTGTAA TA -            #CTTCAATT   1080                                                                 - - AATTTTTTAT GTTTTCATGT CTCTTTATCC ACTCCCTTTA TTTTTACATT AT -            #AATACCAC   1140                                                                 - - ATTCCTCCAA TACTATAATT CTTAAGATAT ATGTGAACAT TAATATCTAA TG -            #ATACATAA   1200                                                                 - - GGTAAGTTGT AAATATTCAT AGAAAAAATA AAATGACTTT TCAAGAAAAC CA -            #ACAACTAA   1260                                                                 - - ATATAAAATA TAGAAAAGTT ATTTACAATT TTGTCCGTTA ACATGTCCAG AT -            #ATTACACT   1320                                                                 - - CTCAAAAGAA AAAGTGTTAG AAAAATCATA TAAAATAGAG TTCAAATTCT TT -            #GTTAGATT   1380                                                                 - - TTTTTTACTG AACATTTAAA ATATATATTG ATATTGATTA TTCATTTTTA TA -            #AATATATT   1440                                                                 - - TTAAAATTAA CATTCAATAT ATATATTTTA AAATTAACAT TCAATATATA TA -            #TTTTAAAG   1500                                                                 - - ACACAGAAGA AACAACAAAT TCCATAAAAT TGTGAGATAA TATTTAACCC TA -            #ACTTTCTT   1560                                                                 - - ATGAACTGAG AGATTTTACA TTTATGAGAA ATGATTGTCC TGTGTTAATT AT -            #CCATGTCA   1620                                                                 - - GCTACCTAAT CACTAGAAAA GCTAATCAGA ATTC       - #                  -     #      1654                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:21:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                              - - GAAGTAATAC TCAAATCTTA TTCCTAGTG         - #                  - #                29                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:22:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                              - - CCCATTTTTC TTTTGTGTTT CAACTTGTGC         - #                  - #               30                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:23:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                              - - ACAAGCAATC AAGAATTAAG TTCAAGAGTC         - #                  - #               30                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:24:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                              - - TAATAATGTT CATTGTATTT GAGAACTGGT         - #                  - #               30                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:25:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                              - - GATTACTAAT AAATCATGTT CTATCCCTAG         - #                  - #               30                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:26:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                              - - AAAGTGTTTG AATAGTAGGA CTAGATCACA         - #                  - #               30                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:27:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1744 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                              - - AAGCTTCAAG TAAGTCTCTG TGATATGTAT GCAAGGGTTC GAAATGAGAA GA -             #AGGCCCTT     60                                                                 - - CAAATTCTAG GTGTACTGGA ATCTAGGAAG GATGAATTAG GAAAAGCTGA TT -            #TTGAGAGA    120                                                                 - - ATTATAAGTG GCCTTATTGA TGGTGGGTTT CGGCAAGATG CCCAACGAAT AT -            #GTGGGATC    180                                                                 - - ATGGAGGCGC AGGATTTCGA TGCATCAAAG GTTAAGGTCA ACCTTATGAA GC -            #CTGTCTCT    240                                                                 - - AGAGGACCTC GTATGAGATA GTTTAGTGGT CATGAATTGG GACATTTTAG TC -            #TTTCTCTG    300                                                                 - - CAAGTGAGTT ACAAATGTAT TACCTTATAT AGGAAGCAAT GTCTGCATGA TT -            #TATCATAC    360                                                                 - - CATGTAACAA ATAAGAATGA ATTTGTTTAT GGATTTTTCC ATTGCTCAGA TT -            #CTGAATTT    420                                                                 - - ACGCAATTTT TTTTTTCTTT TGAACTTTAG TTGTTTGTAT ATACAAATGT CT -            #TCTGTGGC    480                                                                 - - ATGTTCATGG AATTTTCATT TCCAATTATT CAATATTCTT GTGGTGTGAT CA -            #TCACTTTT    540                                                                 - - GTTAGGCAAA TCTGACAGCA CTGATGCCCC CTATCAGGAT TTTTAAACTT GT -            #ATGCGGTA    600                                                                 - - TACTATACTG ATCACAAGAT ACAAACTAAT ATAAATGGAT AGGAAATGCA GA -            #TGGGATGG    660                                                                 - - TTCAAGCTAG TCTTTAATAT TGAGATAGTA CAGAAAATGC AATGCCCAAA GT -            #AAACAACG    720                                                                 - - CTGATATTTC AAAATCACAT ATTAAAGCTA AAGTTGGTAG CAACTAGCGT GA -            #GAGCATCC    780                                                                 - - TAGTCTAGAC TGTGAATGCA GTATTTATAC ACTACAATGA TCTAAATAAG AT -            #GCTACTAA    840                                                                 - - TGCAATCATG CTTAATGTAA TATGAATTGA TCTAAAGTAG CTTGCAAATT TG -            #CTTTGTTT    900                                                                 - - CCGACATACA CTTCAATATG AAAAAAAAAA AAAACACTTT GAGAACTTTT TA -            #AAAAGTAT    960                                                                 - - TAAGTAGGAT TTGACGGCAG AATTTTGTTT CCATATTTAG TTGAAAATAC AT -            #ACAAAACG   1020                                                                 - - TATTTGAAAG TTATATCCGA TTGAATTTGG TTTTAACATA GAAAAAATTC AA -            #CCAAATTA   1080                                                                 - - AGTCCATACT TAAGCATTAA TATAAATATT TCAGTTATTC GACTTCGGTT TC -            #ACGTCTTG   1140                                                                 - - CCATTGTTTT ACATGTGTAA TACTTCAATT AATTTTTTAT GTTTTCATGT CT -            #CTTTATCC   1200                                                                 - - ACTCCCTTTA TTTTTACATT ATAATACCAC ATTCCTCCAA TACTATAATT CT -            #TAAGATAT   1260                                                                 - - ATGTGAACAT TAATATCTAA TGATACATAA GGTAAGTTGT AAATATTCAT AG -            #AAAAAATA   1320                                                                 - - AAATGACTTT TCAAGAAAAC CAACAACTAA ATATAAAATA TAGAAAAGTT AT -            #TTACAATT   1380                                                                 - - TTGTCCGTTA ACATGTCCAG ATATTACACT CTCAAAAGAA AAAGTGTTAG AA -            #AAATCATA   1440                                                                 - - TAAAATAGAG TTCAAATTCT TTGTTAGATT TTTTTTACTG AACATTTAAA AT -            #ATATATTG   1500                                                                 - - ATATTGATTA TTCATTTTTA TAAATATATT TTAAAATTAA CATTCAATAT AT -            #ATATTTTA   1560                                                                 - - AAATTAACAT TCAATATATA TATTTTAAAG ACACAGAAGA AACAACAAAT TC -            #CATAAAAT   1620                                                                 - - TGTGAGATAA TATTTAACCC TAACTTTCTT ATGAACTGAG AGATTTTACA TT -            #TATGAGAA   1680                                                                 - - ATGATTGTCC TGTGTTAATT ATCCATGTCA GCTACCTAAT CACTAGAAAA GC -            #TAATCAGA   1740                                                                 - - ATTC                 - #                  - #                  - #               1744                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:28:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                              - - Asp Glu Ile Asp Phe Glu Leu Gly                                          1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:29:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                              - - GGAAGCTTGA ATTCTGTGAT CTAGTCCTA         - #                  - #                29                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:30:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                              - - GGTCTAGATC CAAAGGGTAT ATATAGGCA         - #                  - #                29                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:31:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                              - - ATCCACTGGT CTCCGTGGGG CGCACAGAG         - #                  - #                29                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:32:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                              - - AACTTTCTCA CTGCATTCAT CATTTACCAG         - #                  - #               30                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:33:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                              - - AGGCTAGCGA AACCAGAACA AGACACAAAA         - #                  - #               30                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:34:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                              - - CATACTTTAT TCCTACCATT ATTTATTACC         - #                  - #               30                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:35:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                              - - AATTTGGTCT CAACTGCAGT TCGTCAACCC G        - #                  - #              31                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:36:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                              - - AATTCGGGTT GACGAACTGC AGTTGAGACC A        - #                  - #              31                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:37:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                              - - ATCCACTGGC CTCCTCGGGT TGGCACAAAC         - #                  - #               30                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:38:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                              - - CTTTTACCAG ATTGTTGTCA CTTTCATGTA         - #                  - #               30                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:39:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                              - - CTGCAGAGGC CAGCGATGCC AATATCAGAG         - #                  - #               30                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:40:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                              - - TCTATCCATT GCCACTTTAT TTATGAATTG         - #                  - #               30                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:41:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                              - - ATTGGATGCA GAGGCAATCA GAAAATACCC         - #                  - #               30                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:42:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                              - - CTACAGAGTG CAACACTGCC TAATTTTTCT         - #                  - #               30                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:43:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                              - - CAATGCATGT TAACAGTAAA AGTCTTGAAA         - #                  - #               30                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:44:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                              - - CTCCACTTTC TTTTTTATTT CTTCTGTTGT         - #                  - #               30                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:45:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                              - - GGATACCCAC AAAATACAAC AAGTGACAAA         - #                  - #               30                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:46:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                              - - CAAAGTGCTT TTAATTGAGC TGTATTTCCC         - #                  - #               30                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:47:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                              - - AAAACTCCTT TTATGATACC CATGGTGAGA         - #                  - #               30                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:48:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                              - - TTTTTGAGTG TATCATTATT GGTGGAGTCA         - #                  - #               30                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:49:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                              - - ATCAGAGACC AGTGTTTGTG TATTTTTCGC         - #                  - #               30                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:50:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                              - - GATATTATGT ATCTCATGCC AGGCCTTTCA         - #                  - #               30                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:51:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                              - - AGGTACTTGA TGTGGTGACT AGCCCAACTG         - #                  - #               30                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:52:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                              - - GGTCTTCATG ACTCAGCGTG TAACGAGTGA         - #                  - #               30                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:53:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                              - - CTCATAGTTT TTCCAAAAGG GTACATCCAC         - #                  - #               30                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:54:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                              - - TATTGTAATT TATTGCACTA TTTGTTTTCT CTGAA       - #                  -     #       35                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:55:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                              - - GGGATACCCA CAAAGTCCTA GTAATGACAA         - #                  - #               30                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:56:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:                              - - AACCAATACT TATGAGTGTA GCACTATTGA ACAAC       - #                  -     #       35                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:57:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:                              - - ATGTGCATGC TGAAGTGGCC GAAGAGGTAG         - #                  - #               30                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:58:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:                              - - ACTACCACTA GTTGTTGTTG TGCCGCTGGT         - #                  - #               30                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:59:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:                              - - ACGTAGTTCT TGTCGAACGG CACGTCCACC         - #                  - #               30                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:60:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:                              - - CGCTGAGACC TAGTAGTACG AGGAATTTGT         - #                  - #               30                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:61:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:                              - - CGCTGAGACC TAGTAGTACG AGGAATTTGT         - #                  - #               30                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:62:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:                              - - TTGGAGCTCA TTTTAAATAT CTCTGTCCTT         - #                  - #               30                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:63:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:                              - - CCTGTTCAAT TTGTGGTTCC            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:64:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:                              - - TTGTGGTCCA GGTCATGGTA            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:65:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:                              - - GGAAGCTTTT CGAAAATTTA TCATTATTGC         - #                  - #               30                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:66:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:                              - - GGTCTAGATT TCTGTGTTTA TCTGTTTGTG G        - #                  - #              31                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:67:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:                              - - ATGGCATTTT CAAGACTTTT ACTGTT          - #                  - #                  26                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:68:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:                              - - GGGTGGTCAG TCCCTT             - #                  - #                      - #    16                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:69:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:                              - - GGAAGCTTGT TAAACTGATT TAAAAG          - #                  - #                  26                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:70:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:                              - - GGTCTAGAGG TGAGAAAAAC AAATCCAAT         - #                  - #                29                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:71:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:                              - - GGAAGCTTTA TTAGGTCGAG TGAGATGGAT         - #                  - #               30                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:72:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:                              - - ATGGGTATCA TAAAAGGAGT TTT           - #                  - #                    23                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:73:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:                              - - AATCGGATCC CCGGGTGGTC AGTCCCTT         - #                  - #                 28                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:74:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:                              - - GGCTGCAGGT TAGATCCCGA ATAATTATCT TAC       - #                  - #             33                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:75:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:                              - - GGTCTAGAGG TGAAAGTTCT AAAGCTAAGC         - #                  - #               30                                                                    __________________________________________________________________________

What is claimed is:
 1. An isolated plant promoter, which in plants isnatively located upstream of and controls the expression of a geneencoding a polypeptide having endo-xyloglucan transferase activity. 2.The isolated plant promoter of claim 1, wherein said gene encoding apolypeptide having endo-xyloglucan transferase activity originates inazuki bean (Vigna angularis).
 3. The isolated plant promoter of claim 1,wherein said gene encoding a polypeptide having endo-xyloglucantransferase activity originates in tomato (Lycopersicon esculentum). 4.The isolated plant promoter of claim 1, wherein said gene encodingpolypeptide having endo-xyloglucan transferase activity originates intobacco (Nicotiana tabacum).
 5. The isolated plant promoter of claim 1,wherein said gene encoding a polypeptide having endo-xyloglucantransferase activity originates in wheat (Triticum asestivum).
 6. Theisolated plant promoter of claim 1, wherein the promoter is contained inany one nucleotide sequence selected from SEQ ID NOs:1, 2, 3, 4, 5, 6,7, and
 8. 7. An isolated DNA molecule comprising the plant promoter ofclaim 1, which is ligated to a useful gene in a state capable ofexpressing the useful gene.
 8. The isolated DNA molecule of claim 7,wherein the useful gene is a gene encoding a protein.
 9. The isolatedDNA molecule of claim 7, wherein the useful gene is a gene encodingantisense RNA.
 10. The isolated DNA molecule of claim 7, wherein theuseful gene is a gene encoding a decoy.
 11. The isolated DNA molecule ofclaim 7, wherein the useful gene is a gene encoding a ribozyme.
 12. Aplant into which the isolated DNA molecule of claim 7 is transferred.13. Plant cells into which the isolated DNA molecule of claim 7 istransferred.
 14. A transgenic plant regenerated from plant cells intowhich the isolated DNA molecule of claim 7 is transferred.
 15. A vectorcomprising the plant promoter of claim
 1. 16. A vector comprising theDNA fragment of claim
 7. 17. A vector of claim 15 which is a plasmidvector.
 18. A vector of claim or 15 which is a viral vector.
 19. A planttransformed with the vector of claim
 15. 20. Plant cells transformedwith the vector of claim
 15. 21. A transgenic plant regenerated from theplant cells of claim
 20. 22. A seed obtained from the plant of any oneof claims 12, 14, 19 or
 21. 23. A vector comprising the plant promoterof claim
 2. 24. A vector comprising the plant promoter of claim
 3. 25. Avector comprising the plant promoter of claim
 4. 26. A vector comprisingthe plant promoter of claim
 5. 27. A vector comprising the plantpromoter of claim 6.